JP2024504694A - Modified double-stranded oligonucleotide - Google Patents

Abstract

One aspect of the invention relates to double-stranded RNA (dsRNA) agents that can inhibit expression of target genes. Another aspect of the invention provides pharmaceutical compositions containing those dsRNA molecules that are suitable for therapeutic use, and for controlling the expression of target genes by administering those dsRNA molecules, e.g., to treat various disease conditions. Concerning methods of inhibiting. TIFF2024504694000123.tif92156

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is filed under 35 U.S.C. Provisional Patent Application No. 63/146,115, U.S. Provisional Patent Application No. 63/148,991 filed February 12, 2021, U.S. Provisional Patent Application No. 63/153,983 filed February 26, 2021, 2021 U.S. Provisional Patent Application No. 63/156,476 filed on March 4, 2021, U.S. Provisional Patent Application No. 63/161,313 filed on March 15, 2021, U.S. Provisional Patent Application No. 63/164,467, U.S. Provisional Patent Application No. 63/179,607 filed April 26, 2021, and U.S. Provisional Patent Application No. 63/141,748 filed April 29, 2021. The contents of each of these US provisional patent applications are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION The present invention relates to dsRNA molecules having specific motifs advantageous for inhibition of target gene expression, and dsRNA agent compositions suitable for therapeutic use. In addition, the present invention provides methods of inhibiting target gene expression by administering these dsRNA agents, eg, for the treatment of various diseases.

background
RNA interference, or "RNAi," is a term first coined by Fire and colleagues to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Fire et al. (1998) Nature 391,806-811, Elbashir et al. (2001) Genes Dev.15,188-200 (Non-Patent Document 1)). Short dsRNA directs gene-specific post-transcriptional silencing in many organisms, including vertebrates, and has become a new tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. Although RISC is known to contain short RNAs (approximately 22 nucleotides) derived from double-stranded RNA triggers, the protein component of this activity remained unknown.

There remains a need in the art for effective nucleotide motifs or chemical motifs for dsRNA molecules that are advantageous in inhibiting target gene expression. The present invention is directed to that effort.

Fire et al. (1998) Nature 391,806-811, Elbashir et al. (2001) Genes Dev.15,188-200

SUMMARY The present invention provides effective nucleotide motifs or chemical motifs for dsRNA molecules that are advantageous for inhibition of target gene expression, and RNAi compositions suitable for therapeutic use.

In particular, the inventors have demonstrated that double-stranded RNA (dsRNA) molecules having a 2'-fluoronucleotide at least in position 10 of the sense strand unexpectedly and surprisingly exhibit improved in vitro potency, i.e., increased RNA It was discovered that it has interference (RNAi) activity. Thus, in one aspect, herein is a double-stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; Double-stranded RNA (dsRNA) is provided, the strand comprising a 2'-fluoronucleotide at position 10 counting from the 5' end of the sense strand.

Note that the sense strand may further comprise one or more, for example 1, 2, 3, 4 or 5, additional 2'-fluoronucleotides. Thus, in some embodiments, the sense strand includes 1, 2, 3, 4 or 5 additional 2'-fluoronucleotides. Additional 2'-fluoronucleotides can be located anywhere in the sense strand. Thus, in some embodiments, the sense strand further comprises a 2'-fluoronucleotide at position 8, 9, 11 or 12 counting from the 5' end of the sense strand. For example, the sense strand further includes a 2'-fluoronucleotide at position 9 counting from the 5' end of the sense strand. In other words, the sense strand contains 2'-fluoronucleotides at positions 9 and 10 counting from the 5' end of the sense strand. In another example, the sense strand further comprises a 2'-fluoronucleotide at position 11 counting from the 5' end of the sense strand. For example, the sense strand contains 2'-fluoronucleotides at positions 10 and 11 counting from the 5' end of the sense strand.

In some embodiments, the sense strand includes 2'-fluoronucleotides at positions 9, 10, and 11 counting from the 5' end of the sense strand. In some other embodiments, the sense strand includes 2'-fluoronucleotides at positions 8, 9, and 10 counting from the 5' end of the sense strand. In yet some other embodiments, the sense strand comprises 2'-fluoronucleotides at positions 10, 11 and 12 counting from the 5' end of the sense strand.

In some embodiments of any one of the above aspects, the sense strand does not include a 2'-fluoronucleotide at position 7 counting from the 5' end of the sense strand. For example, the sense strand contains a 2'-OMe nucleotide at position 7 counting from the 5' end of the sense strand.

The antisense strand of a dsRNA molecule described herein can include one or more 2'-deoxy, eg, 2'-H, nucleotides. For example, the antisense strand includes 1, 2, 3, 4, 5, 6 or more 2'-deoxynucleotides. In some embodiments, the antisense strand comprises 2, 3, 4, 5 or 6 2'-deoxynucleotides. The 2'-deoxynucleotide can be located anywhere in the antisense strand. For example, the antisense strand may contain 2'-deoxynucleotides at 1, 2, 3, 4, 5, or 6 of positions 2, 5, 7, 12, 14, and 16 counting from the 5' end of the antisense strand. including. In some embodiments, the antisense comprises 2'-deoxynucleotides at positions 2 and 12 counting from the 5' end of the antisense strand. In some embodiments, the antisense comprises 2'-deoxynucleotides at positions 5 and 7 counting from the 5' end of the antisense strand. In some embodiments, the antisense comprises 2'-deoxynucleotides at positions 2, 5, 7, and 12 counting from the 5' end of the antisense strand.

In some embodiments, the antisense can include one or more, eg, 1, 2, 3, 4, 5 or more, 2'-fluoronucleotides. For example, the antisense strand can include a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand.

In some embodiments, the antisense strand comprises a 2'-fluoronucleotide at position 14, counting from the 5' end of the antisense strand, and a nucleotide other than 2'-deoxy or 2'-fluoro at position 16. include. For example, the antisense strand contains a 2'-fluoronucleotide at position 14, counting from the 5' end of the antisense strand, and a nucleotide other than 2'-deoxy or 2'-fluoro at position 16.

In some embodiments, the antisense strand includes 2'-deoxynucleotides at positions 2 and 12, counting from the 5' end of the antisense strand, and a 2'-fluoronucleotide at position 14. In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2 and 12, counting from the 5' end of the antisense strand, a 2'-fluoronucleotide at position 14, and a 2'-fluoronucleotide at position 16. Contains nucleotides other than '-deoxy or 2'-fluoro. For example, the antisense strand contains 2'-deoxynucleotides at positions 2 and 12, counting from the 5' end of the antisense strand, a 2'-fluoronucleotide at position 14, and a 2'-OMe nucleotide at position 16. include.

In some embodiments, the antisense strand includes a 2'-deoxynucleotide at position 14 counting from the 5' end of the antisense strand, and the sense strand includes a 2'-deoxynucleotide at position 7 counting from the 5' end of the sense strand; Contains nucleotides other than 2'-fluoro. For example, the antisense strand contains 2'-deoxynucleotides at positions 2, 12, and 14 counting from the 5' end of the antisense strand, and the sense strand contains 2'-deoxynucleotides at position 10 counting from the 5' end of the sense strand. - contains a fluoronucleotide and contains a nucleotide other than 2'-fluoro at position 7.

In various embodiments, the dsRNA molecule has a double-stranded (duplex) region of 19-25 base pairs. For example, a dsRNA molecule has a double-stranded region of 20, 21, 22, 23 or 24 base pairs. In some particular embodiments, the dsRNA molecule has a double-stranded (duplex) region of 20, 21 or 22 base pairs.

In some embodiments, the dsRNA molecule includes a ligand. For example, the sense strand of a dsRNA molecule includes a ligand. Exemplary ligands include, but are not limited to, ASGPR ligands and ligands containing lipophilic groups.

A dsRNA molecule can include one or more, eg, 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate linkages. The phosphorothioate linkage can be present on only one of the strands of the dsRNA or on both strands. For example, the sense strand can contain 1, 2, 3 or 4 phosphorothioate linkages. In another non-limiting example, the antisense strand can include 1, 2, 3, 4, 5 or 6 phosphorothioate linkages. In some embodiments, the sense strand comprises 1, 2, 3 or 4 phosphorothioate linkages and the antisense independently comprises 1, 2, 3, 4, 5 or 6 phosphorothioate linkages. For example, the sense strand contains 1 or 2 phosphorothioate linkages and the antisense strand contains 1, 2, 3 or 4 phosphorothioate linkages.

In some embodiments, the sense strand comprises at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 5' end of the sense strand, and the antisense strand comprises at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 5' end of the antisense strand. antisense contains at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 3' end of the antisense strand; Including further. For example, the sense strand contains phosphorothioate linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, counting from the 5' end of the sense strand, and the antisense strand contains phosphorothioate linkages, counting from the 5' end of the antisense strand. It contains phosphorothioate linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, and between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 3' end of the antisense strand.

In some embodiments, the remaining nucleotides in the dsRNA are 2'-OMe nucleotides. For example, all remaining nucleotides in the sense strand are 2'-OMe nucleotides. In other words, only the sense strand contains 2'-fluoronucleotides and 2'-OMe nucleotides.

It is understood that the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference. In other words, the dsRNA molecules of the invention are capable of inhibiting target gene expression.

In another aspect, the invention further provides methods for delivering a dsRNA molecule of the invention to a specific target in a subject by subcutaneous or intravenous administration. The invention further provides a dsRNA molecule of the invention for use in a method for delivering the agent to a specific target in a subject by subcutaneous or intravenous administration.

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fee.

Figures 1A and 1B demonstrate that dsRNA according to exemplary embodiments of the invention has improved in vitro potency compared to parent dsRNA molecules when administered at 10 nM (Figure 1A) or 1 nM (Figure 1). This is a graph showing. Figures 2A-2D are graphs showing that dsRNA molecules according to embodiments of the invention have improved in vivo efficacy compared to the parent molecule. The parental duplexes were AD-1181401 (Sequence 1, Figure 2A), AD-1181410 (Sequence 2, Figure 2B), AD-1181426 (Sequence 3, Figure 2C) and AD-1181451 (Sequence 4, Figure 2D). be. Figures 3A-3D show how dsRNAs according to exemplary embodiments of the invention targeting various targets were compared to parent dsRNA molecules when administered at 1 nM (Figures 3A and 3B) or 0.1 nM (Figure 3C). 2 is a graph showing improved in vitro efficacy. Figures 4A-4C are graphs showing that the presence of a 2'-fluoronucleotide at position 10 of the sense strand, counting from the 5' end of the sense strand, enhances the RNAi efficacy of dsRNA molecules compared to the parent. . 1 is a schematic representation of several exemplary designs of dsRNA molecules according to embodiments of the invention. 1 is a schematic representation of several exemplary designs of dsRNA molecules according to embodiments of the invention. FIGS. 6A and 6B are graphs showing in vitro target knockdown (FIG. 6A) and log2 activity (FIG. 6B) of exemplary dsRNAs according to some embodiments of the present disclosure compared to exemplary parent dsRNA molecules. Figures 7A and 7B show in vitro targeted AGT knockdown (Figure 6A) and log2 activity (Figure 6B) of exemplary dsRNAs according to some embodiments of the present disclosure compared to exemplary parent dsRNA molecules that target AGT. This is a graph showing. Sense strands of exemplary dsRNA molecules in mouse liver homogenates (Figures 8A and 8E), rat liver homogenates (Figures 8B and 8F), rat brain homogenates (Figures 8C and 8G), and cynomolgus liver homogenates (Figures 8D and 8H). 8A-8D) and the antisense strand (FIGS. 8E-8H), demonstrating similar or improved metabolic stability. Parent duplexes are AD-1181401 (TTR Seq 1), AD-1181410 (TTR Seq 2) and AD-74210 (F12). See description of Figure 8A. See description of Figure 8A. See description of Figure 8A. See description of Figure 8A. See description of Figure 8A. See description of Figure 8A. See description of Figure 8A. Figures 9A and 9B demonstrate similar or improved metabolic stability of exemplary dsRNA molecules in mice. Parent duplexes are AD-1181401 (TTR Seq 1), AD-1181410 (TTR Seq 2) and AD-74210 (F12). FIGS. 10A-10D show that dsRNA molecules according to embodiments of the invention were tested in non-human primates, mice (FIGS. 10A and 10B) and cynomolgus monkeys (FIGS. 10C and 10D), with parent molecules AD-74210 (FIGS. 10A and 10C) and AD -75885 (FIGS. 10B and 10D) has improved in vivo efficacy and/or duration.

DETAILED DESCRIPTION In one aspect, the invention provides double-stranded RNA (dsRNA) agents capable of inhibiting target gene expression. Without limitation, the dsRNA agents of the invention can be used in place of dsRNA molecules for use in RNA interference-based gene silencing techniques, including but not limited to in vitro or in vivo applications. can do.

Generally, a dsRNA molecule includes a sense strand (also called a passenger strand) and an antisense strand (also called a guide strand). Each strand of a dsRNA molecule can range in length from 15 to 35 nucleotides. For example, each strand may be 17-35 nucleotides long, 17-30 nucleotides long, 25-35 nucleotides long, 27-30 nucleotides long, 17-23 nucleotides long, 17-21 nucleotides long, 17-19 nucleotides long, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. Without limitation, the sense and antisense strands may be of equal or unequal length. For example, the sense and antisense strands independently have a length of 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

In some embodiments, the antisense strand is 15-35 nucleotides in length. In some embodiments, the antisense strand is 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23 , 19-21, 21-25, 21-25 or 21-23 nucleotides in length. For example, the antisense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides long. Something can happen. In some embodiments, the antisense strand is 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, the antisense strand is 21, 22, 23, 24 or 25 nucleotides long. In some particular embodiments, the antisense strand is 22, 23 or 24 nucleotides long. For example, the antisense strand is 23 nucleotides long.

Like the antisense strand, the sense strand can be 15-35 nucleotides in length in some embodiments. In some embodiments, the sense strand is 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25 or 21-23 nucleotides in length. For example, the sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides long. be able to. In some embodiments, the sense strand is 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, the sense strand is 19, 20, 21, 22 or 23 nucleotides long. In some particular embodiments, the sense strand is 20, 21 or 22 nucleotides long. For example, the sense strand is 21 nucleotides long.

In some embodiments, the sense strand can be 15-35 nucleotides long, and the antisense strand can be 15-35 nucleotides long, independently of the sense strand. In some embodiments, the sense strand is 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25 or 21-23 nucleotides in length, and the antisense strands are independently 15-35, 17-35, 17-30, 25-35, 27-30, 17 ~23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25 or 21-23 nucleotides long. For example, the sense strand and antisense strand independently , 34 or 35 nucleotides in length. In some embodiments, the sense and antisense strands can independently be 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, the sense strand is 19, 20, 21, 22 or 23 nucleotides long and the antisense strand is 21, 22, 23, 24 or 25 nucleotides long. In some particular embodiments, the sense strand is 20, 21 or 22 nucleotides long and the antisense strand is 22, 23 or 24 nucleotides long. For example, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

The sense and antisense strands typically form a double-stranded region or a double-stranded region. Without limitation, the duplex region of the dsRNA agents described herein can be 12 to 35 nucleotides (or bases) pairs in length. For example, the duplex region may be 14-35 nucleotide pairs long, 17-30 nucleotide pairs long, 25-35 nucleotide pairs long, 27-35 nucleotide pairs long, 17-23 nucleotide pairs long, 17-21 nucleotide pairs long, 17-35 nucleotide pairs long, It can be 19 nucleotide pairs long, 19-25 nucleotide pairs long, 19-23 nucleotide pairs long, 19-21 nucleotide pairs long, 21-25 nucleotide pairs long, or 21-23 nucleotide pairs long. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 nucleotide pairs in length. In some embodiments, the duplex region is 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs in length. For example, the duplex region is 19, 20, 21, 22 or 23 nucleotide pairs long. In some embodiments, the duplex region is 20, 21 or 22 nucleotide pairs long. For example, a dsRNA molecule has a 21 base pair duplex region.

As described herein, the dsRNA molecules of the invention include at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, 2'-deoxy, For example, it can further include a 2'-H nucleotide. For example, a dsRNA can contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-deoxy, eg, 2'-H, nucleotides. The 2'-deoxynucleotide may be present at any nucleotide position on the sense or antisense strand, or on both strands. In one non-limiting example, the sense strand does not include a 2'-deoxynucleotide at position 11 counting from the 5' end of the sense strand.

In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5 or 6 2'-deoxynucleotides. For example, the antisense strand can contain 2, 3, 4, 5 or 6 2'-deoxynucleotides. The 2'-deoxynucleotide can be located anywhere in the antisense strand. For example, the antisense strand may contain 2'-deoxynucleotides at 1, 2, 3, 4, 5, or 6 of positions 2, 5, 7, 12, 14, and 16 counting from the 5' end of the antisense strand. including. In one non-limiting example, the antisense strand comprises 2'-deoxynucleotides in 1, 2, 3 or 4 of positions 2, 5, 7 and 12 counting from the 5' end of the antisense strand. .

In some embodiments, the antisense comprises 2'-deoxynucleotides at positions 5 and 7 counting from the 5' end of the antisense strand. For example, the antisense strand contains 2'-deoxynucleotides at positions 5, 7, and 12 counting from the 5' end of the antisense strand. In some embodiments, the antisense strand includes 2'-deoxynucleotides at positions 2, 5, and 7 counting from the 5' end of the antisense strand. For example, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, and 12 counting from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12 and 14 counting from the 5' end of the antisense strand. For example, the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, 14 and 16 counting from the 5' end of the antisense strand.

In some embodiments, the antisense comprises a 2'-deoxynucleotide at position 2 or 12 counting from the 5' end of the antisense strand. For example, antisense contains a 2'-deoxynucleotide at position 12 counting from the 5' end of the antisense strand.

In some embodiments, the sense strand comprises a 2'-fluoronucleotide at position 10, counting from the 5' end of the sense strand, and the antisense comprises a 2'-fluoronucleotide at positions 5 and 7, counting from the 5' end of the antisense strand. Contains deoxynucleotides. For example, the sense strand contains 2'-fluoronucleotides at positions 9 and 10 counting from the 5' end of the sense strand, and the antisense strand contains 2'-deoxynucleotides at positions 5 and 7 counting from the 5' end of the antisense strand. including. In another example, the sense strand includes 2'-fluoronucleotides at positions 8, 9, and 10, counting from the 5' end of the sense strand, and the antisense 2'-fluoronucleotides at positions 5 and 7, counting from the 5' end of the antisense strand. Contains 2'-deoxynucleotides.

In some embodiments, the sense strand comprises 2'-fluoronucleotides at positions 10 and 11, counting from the 5' end of the sense strand, and the antisense comprises 2'-fluoronucleotides at positions 5 and 7, counting from the 5' end of the antisense strand. '-Contains deoxynucleotides. For example, the sense strand contains 2'-fluoronucleotides at positions 10, 11, and 12, counting from the 5' end of the sense strand, and the antisense contains 2'-fluoronucleotides at positions 5 and 7, counting from the 5' end of the antisense strand. Contains deoxynucleotides.

In some embodiments of any one of the above aspects, the sense strand does not include a 2'-fluoronucleotide at position 7 counting from the 5' end of the sense strand. For example, the sense strand contains a 2'-fluoronucleotide in at least one, e.g. '-Free of fluoronucleotides. In some embodiments, the sense strand includes a 2'-fluoronucleotide at position 10, counting from the 5' end of the sense strand, and a nucleotide other than a 2'-fluoronucleotide at position 7. For example, the sense strand contains a 2'-fluoronucleotide at position 10 and a 2'-OMe nucleotide at position 7 counting from the 5' end of the sense strand.

In some embodiments, the sense strand includes 2'-fluoronucleotides at positions 9 and 10, counting from the 5' end of the sense strand, and a nucleotide other than a 2'-fluoronucleotide at position 7. For example, the sense strand contains 2'-fluoronucleotides at positions 9 and 10, counting from the 5' end of the sense strand, and a 2'-OMe nucleotide at position 7.

In some embodiments, the sense strand includes 2'-fluoronucleotides at positions 10 and 11, counting from the 5' end of the sense strand, and a nucleotide other than a 2'-fluoronucleotide at position 7. For example, the sense strand contains 2'-fluoronucleotides at positions 10 and 11, counting from the 5' end of the sense strand, and a 2'-OMe nucleotide at position 7.

In some embodiments, the sense strand includes 2'-fluoronucleotides at positions 9, 10, and 11 counting from the 5' end of the sense strand, and a nucleotide other than a 2'-fluoronucleotide at position 7. For example, the sense strand contains 2'-fluoronucleotides at positions 9, 10 and 11 counting from the 5' end of the sense strand and a 2'-OMe nucleotide at position 7.

In some embodiments, the sense strand comprises 2'-fluoronucleotides at positions 9, 10 and 11 counting from the 5' end of the sense strand, and the remaining nucleotides in the sense strand are 2'-OMe nucleotides.

2'-fluoro modification (antisense strand)
A dsRNA molecule of the invention comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. Without limitation, all 2'-fluoronucleotides can be present in one strand. In some embodiments, both the sense and antisense strands include at least one 2'-fluoronucleotide. The 2'-fluoro modification can be present on any nucleotide of the sense or antisense strand. For example, a 2'-fluoro modification can be present on every nucleotide on the sense strand and/or on the antisense strand, or each 2'-fluoro modification can be present on the sense or antisense strand in an alternating pattern. or the sense or antisense strand can contain both 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand may be the same as the 2'-fluoro modifications on the antisense strand. There may be shifts relative to the alternating pattern of fluoromodification.

In some embodiments, the antisense strand comprises at least two (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. Without limitation, the 2'-fluoro modification in the antisense strand can be present at any position.

In some embodiments, the antisense comprises one or more, eg, 1, 2, 3, 4, 5 or more, 2'-fluoronucleotides. For example, the antisense strand includes 1, 2, 3, 4 or 5 or more 2'-fluoronucleotides. In some embodiments, the antisense strand includes 1, 2 or 3 2'-fluoronucleotides. For example, the antisense strand contains a single 2'-fluoronucleotide. Note that the 2'-fluoronucleotide can be located anywhere in the antisense strand. For example, the 2'-fluoronucleotide can be at position 2 or 14 of the antisense strand, counting from the 5' end. In some embodiments, the antisense comprises a 2'-fluoronucleotide at position 14 of the antisense strand, counting from the 5' end.

In some embodiments, the antisense comprises a 2'-fluoronucleotide at position 14 and 2'-deoxynucleotides at positions 5 and 7 counting from the 5' end of the antisense strand. For example, an antisense contains a 2'-fluoronucleotide at position 14 and 2'-deoxynucleotides at positions 5, 7, and 12 counting from the 5' end of the antisense strand. In a further example, the antisense comprises a 2'-fluoronucleotide at position 14 and 2'-deoxynucleotides at positions 2, 5, 7 and 12 counting from the 5' end of the antisense strand.

In some embodiments, the antisense strand includes 2'-deoxynucleotides at positions 2 and 12 counting from the 5' end of the antisense strand. In some embodiments, the antisense strand further comprises a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand. For example, the antisense strand contains 2'-deoxynucleotides at positions 2 and 12, counting from the 5' end of the antisense strand, and a 2'-fluoronucleotide at position 14.

In some embodiments, the antisense strand comprises a nucleotide other than a 2'-deoxynucleotide at position 16 counting from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises a nucleotide other than a 2'-fluoronucleotide at position 16 counting from the 5' end of the antisense strand. For example, the antisense strand contains 2'-deoxynucleotides at positions 2 and 12, counting from the 5' end of the antisense strand, and a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16. In some embodiments, the antisense strand includes 2'-deoxynucleotides at positions 2 and 12 and a 2'-OMe nucleotide at position 16, counting from the 5' end of the antisense strand.

In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2 and 12, counting from the 5' end of the antisense strand, a 2'-fluoronucleotide at position 14, and a 2'-fluoronucleotide at position 16. Contains nucleotides other than '-deoxy. In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2 and 12, counting from the 5' end of the antisense strand, a 2'-fluoronucleotide at position 14, and a 2'-fluoronucleotide at position 16. '-Contains nucleotides other than fluoro. For example, the antisense strand contains 2'-deoxynucleotides at positions 2 and 12, counting from the 5' end of the antisense strand, a 2'-fluoronucleotide at position 14, and a 2'-deoxy or 2'-fluoronucleotide at position 16. '-Contains nucleotides other than fluoronucleotides. In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2 and 12, counting from the 5' end of the antisense strand, a 2'-fluoronucleotide at position 14, and a 2'-fluoronucleotide at position 16. '-OMe contains nucleotides.

In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, and 12 counting from the 5' end of the antisense strand, and a 2'-fluoronucleotide at position 14, The remaining nucleotides in the antisense strand are 2'-OMe nucleotides.

In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, and 12 counting from the 5' end of the antisense strand, and a 2'-fluoronucleotide at position 14, and The sense strand contains 2'-fluoronucleotides at positions 9, 10 and 11 counting from the 5' end of the sense strand, and the remaining nucleotides in the antisense and sense strands are 2'-OMe nucleotides.

In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12, and 14 counting from the 5' end of the antisense strand, and the remaining nucleotides in the antisense strand are 2'-deoxynucleotides. '-OMe nucleotide.

In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12, and 14 counting from the 5' end of the antisense strand, and the sense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12, and 14 counting from the 5' end of the sense strand; It contains 2'-fluoronucleotides at positions 9, 10 and 11, counting from the end, and the remaining nucleotides in the antisense and sense strands are 2'-OMe nucleotides.

In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12, and 16 counting from the 5' end of the antisense strand, and the remaining nucleotides in the antisense strand are 2'-deoxynucleotides. '-OMe nucleotide.

In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12, and 16 counting from the 5' end of the antisense strand, and the sense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12, and 16 counting from the 5' end of the It contains 2'-fluoronucleotides at positions 9, 10 and 11 counting from , and the remaining nucleotides in the antisense and sense strands are 2'-OMe nucleotides.

In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12, 14, and 16 counting from the 5' end of the antisense strand, and the remaining nucleotides in the antisense strand is a 2'-OMe nucleotide.

In some embodiments, the antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12, 14, and 16 counting from the 5' end of the antisense strand, and the sense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12, 14, and 16 counting from the 5' end of the It contains 2'-fluoronucleotides at positions 9, 10 and 11, counting from the end, and the remaining nucleotides in the antisense and sense strands are 2'-OMe nucleotides.

The remaining nucleotides, ie, those at positions not explicitly defined in the sense and/or antisense strands, can be unmodified or modified nucleotides. Thus, in some embodiments, the remaining nucleotides, ie, those at positions not explicitly defined in the sense strand, are unmodified or modified nucleotides. For example, the remaining nucleotides, i.e. those in positions not explicitly defined in the sense strand, consist of 2'-OMe, 2'-F, 2'-H and 2'-OC _10-30 aliphatic groups. Optionally, no more than one modified nucleotide is a 2'-OC _10-30 aliphatic group.

In some embodiments, the remaining nucleotides, ie, nucleotides at positions not explicitly defined in the antisense strand, are unmodified or modified nucleotides. For example, the remaining nucleotides, ie, those at positions not explicitly defined in the antisense strand, can be modified nucleotides. In some embodiments, the remaining nucleotides, i.e., nucleotides at positions not explicitly defined in the antisense strand, are from 2'-OMe, 2'-F, 2'-H, GNA, and 3'-RNA. and the 3'-RNA is optionally 3'-OH, provided that no more than one modified nucleotide is GNA or 3'-RNA.

In some embodiments, the remaining nucleotides in the sense and/or antisense strands are 2'-OMe nucleotides.

As described herein, a dsRNA agent comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides containing modified sugars. can be included. "Modified sugar" refers to sugars other than 2'-deoxy (ie, 2'-H), 2'-OH, 2'-F, or 2'-OMe ribose sugars. Some exemplary nucleotides containing modified sugars include locked nucleic acid (LNA), HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-O-N-methylacetamide. (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP) and 2'-ara-F. be. Thus, in some embodiments, the dsRNA agent is an acyclic nucleotide, locked nucleic acid (LNA), HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'- O-N-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP) and 2'- ara-F, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides independently selected from the group consisting of ara-F. can. Nucleotides containing modified sugars can occur anywhere in the dsRNA molecule. For example, nucleotides containing modified sugars can be present in the sense strand, or nucleotides containing modified sugars can be present in the antisense strand. When more than one nucleotide containing a modified sugar is present in a dsRNA molecule, they can all be present in the sense strand, in the antisense strand, or in both the sense and antisense strands.

In some embodiments, unmodified nucleotides are unmodified nucleobases, ie, 2'-OH nucleotides that include adenine, guanine, cytosine or uracil.

In some embodiments, the dsRNA comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides that include a non-natural nucleobase. be able to. "Non-natural nucleobase" means a nucleobase other than adenine, guanine, cytosine, uracil or thymine. Exemplary non-natural nucleobases include inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, and substituted or modified analogs of adenine, guanine, cytosine and uracil, such as 2-amino Adenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6- Azocytosine and 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl, etc. 8-substituted adenine and 8-substituted guanine, 5-trifluoromethyl and other 5-substituted uracil and 5-substituted cytosine, 7-methylguanine, 5-substituted pyrimidine, 6-azapyrimidine, and N-2, N -6 and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkyl Guanine, 5-alkylcytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazole, 2-pyridinone , 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5- Methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, ^N4 -acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyl Examples include, but are not limited to, adenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine, or O-alkylated bases. Additional purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, pages 858-859 of Concise Encyclopedia of Polymer Science and Engineering, edited by Kroschwitz, JI, John Wiley & Sons, 1990; and Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

In some embodiments, the unnatural nucleobase is inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino) ) adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio) ^-N6- (isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7- (deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-( thioalkyl)adenine, 8-(thiol)adenine, N ⁶ -(isopentyl)adenine, N ⁶ -(methyl)adenine, N ⁶ ,N ⁶ -(dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl) Guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine , 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5- (halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, ^N4- (acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4 -(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5 -(Methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-( Aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylamino) alkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5 -(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, ^N3- (methyl)uracil, 5-uracil (i.e. pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil , 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4 -(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2( thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl) -2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkyl) Aminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1- (aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio )-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phentiazin-1-yl, 1-(aza)-2-(thio)-3-( aza)-phentiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3- (aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phentiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3 -(aza)-phentiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1- (aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phentiazin-1-yl , 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phentiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1,3-(diaza) -2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7 -(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phentiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio )-3-(aza)-phentiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2 -Aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyryl, 5-(methyl)isocarbostyryl Lylyl, 3-(methyl)-7-(propynyl)isocarbostyrylyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)- imidizopyridinyl, pyrrolopyridinyl, isocarbostyrylyl, 7-(propynyl)isocarbostyrylyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl) Benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted selected from the group consisting of pyrimidines, ^N2 -substituted purines, ^N6 -substituted purines, ^O6 -substituted purines, substituted 1,2,4-triazoles, and any O-alkylated or N-alkylated derivatives thereof be able to.

Nucleotides containing non-natural nucleobases can be present anywhere in the dsRNA molecule. For example, nucleotides containing a non-natural nucleobase can be present in the sense strand, or nucleotides containing a non-natural nucleobase can be present in the antisense strand. If more than one nucleotide containing a non-natural nucleobase is present in a dsRNA molecule, they can all be present in the sense strand, in the antisense strand, or in both the sense and antisense strands.

The dsRNA molecules of the invention can further include at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate internucleotide linkage modification or the methylphosphonate internucleotide linkage modification can be present at any position on the strand and on any nucleotide of the sense or antisense strand, or both. For example, an internucleotide linkage modification may be present on every nucleotide on the sense strand and/or the antisense strand, or each internucleotide linkage modification may be present in an alternating pattern on the sense or antisense strand. Often, the sense or antisense strand contains both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may be the same as the internucleotide linkage modifications on the antisense strand. It may be shifted with respect to the alternating pattern of part modifications.

In some embodiments, the dsRNA molecule comprises a phosphorothioate internucleotide linkage modification or a methylphosphonate internucleotide linkage modification in the overhang region. For example, an overhang region includes two nucleotides with a phosphorothioate internucleotide linkage or a methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications may be made to link overhanging nucleotides to terminally paired nucleotides within the duplex region. For example, at least two, three or four or all of the overhanging nucleotides may be linked with phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages, optionally with the overhanging nucleotides and the pairs adjacent to the overhanging nucleotides. Additional phosphorothioate or methylphosphonate internucleotide linkages may be present that link the nucleotides. For example, there are at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of those three nucleotides are overhang nucleotides, and the third is a pair of nucleotides adjacent to the overhang nucleotide. It may also be a nucleotide. Preferably, these three terminal nucleotides may be at the 3' end of the antisense strand.

In some embodiments, the sense strand of the dsRNA molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate internucleotide linkages. comprising 1 to 10 blocks of 2 to 10 phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages, separated by 1 to 10 blocks of phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages; is placed anywhere in the oligonucleotide sequence, and the sense strand is an antisense strand containing any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages, and phosphate internucleotide linkages, or phosphorothioate internucleotide linkages. It is paired with an antisense strand that includes a linkage or either a methylphosphonate internucleotide linkage or a phosphate internucleotide linkage.

In some embodiments, the antisense strand of the dsRNA molecule is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 comprising two blocks of two phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages separated by a phosphate internucleotide linkage of is placed anywhere in the oligonucleotide sequence, and the antisense strand is a sense strand that includes any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages, and phosphate internucleotide linkages, or phosphorothioate internucleotide linkages. It is paired with an antisense strand that includes a linkage or either a methylphosphonate internucleotide linkage or a phosphate internucleotide linkage.

In some embodiments, the antisense strand of the dsRNA molecule is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate nucleotides. two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being an oligonucleotide sequence; and the antisense strand includes any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages, and phosphate internucleotide linkages, or phosphorothioate internucleotide linkages or methyl phosphorothioate internucleotide linkages. It is paired with an antisense strand that contains either a phosphonate or a phosphate internucleotide linkage.

In some embodiments, the antisense strands of the dsRNA molecule are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 phosphate internucleotide linkages. two blocks of four phosphorothioate or methylphosphonate internucleotide linkages, one of which can be used at any point in the oligonucleotide sequence. and the antisense strand comprises any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages and phosphate internucleotide linkages, or phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages. The antisense strand is paired with an antisense strand containing either a nucleotide or a phosphate internucleotide linkage.

In some embodiments, the antisense strands of the dsRNA molecule are separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages. two blocks of phosphorothioate or methylphosphonate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being placed at any position in the oligonucleotide sequence. , the antisense strand is a sense strand comprising any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages and phosphate internucleotide linkages, or phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages or phosphate nucleotide linkages. It is paired with an antisense strand that includes either an interlinker.

In some embodiments, the antisense strand of the dsRNA molecule comprises six phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate internucleotide linkages. two blocks of linkages or methylphosphonate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being placed at any position in the oligonucleotide sequence; The strand can be a sense strand that includes any combination of phosphorothioate internucleotide linkages, methylphosphonate internucleotide linkages, and phosphate internucleotide linkages, or a sense strand that contains phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages or phosphate internucleotide linkages. It is paired with an antisense strand containing either.

In some embodiments, the antisense strand of the dsRNA molecule comprises seven phosphorothioate internucleotide linkages or methyl The antisense strand contains two blocks of phosphonate internucleotide linkages, one of the phosphorothioate internucleotide linkages or the methylphosphonate internucleotide linkages placed at any position in the oligonucleotide sequence; a sense strand comprising any combination of internucleotide linkages, methylphosphonate internucleotide linkages and phosphate internucleotide linkages, or comprising either phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages or phosphate internucleotide linkages Pairs with antisense strand.

In some embodiments, the antisense strand of the dsRNA molecule comprises eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages. one of the phosphorothioate internucleotide linkages or the methylphosphonate internucleotide linkages is placed anywhere in the oligonucleotide sequence, and the antisense strand contains two blocks of phosphorothioate internucleotide linkages or methylphosphonate internucleotide linkages; , a sense strand comprising any combination of a methylphosphonate internucleotide linkage and a phosphate internucleotide linkage, or an antisense strand comprising either a phosphorothioate internucleotide linkage or a methylphosphonate internucleotide linkage or a phosphate internucleotide linkage. , are paired.

In some embodiments, the antisense strand of the dsRNA molecule comprises a block of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, or 4 phosphate internucleotide linkages. , one of the phosphorothioate internucleotide linkages or the methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence, and the antisense strand includes a phosphorothioate internucleotide linkage, a methylphosphonate internucleotide linkage, a sense strand containing any combination of an internucleotide linkage and a phosphate internucleotide linkage, or an antisense strand containing either a phosphorothioate internucleotide linkage or a methylphosphonate internucleotide linkage or a phosphate internucleotide linkage; ing.

In some embodiments, the dsRNA molecules of the invention include one or more phosphorothioate internucleotide linkage modifications or methylphosphonate internucleotide linkage modifications within 1 to 10 of the terminal positions of the sense and/or antisense strands. It further includes linkage modification. For example, at one or both ends of the sense and/or antisense strand, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides form a phosphorothioate internucleotide linkage or a methylphosphonate internucleotide linkage. can be connected by

In some embodiments, the dsRNA molecules of the invention contain one or more phosphorothioate internucleotide linkages within 1 to 10 of the internal regions of the duplex of each of the sense and/or antisense strands. modification or methylphosphonate internucleotide linkage modification. For example, in positions 8 to 16 of the duplex region counting from the 5' end of the sense strand, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides are present between the phosphorothioate methylphosphonate nucleotides. Optionally, the dsRNA molecules further include one or more phosphorothioate internucleotide linkage modifications or methylphosphonate internucleotide linkage modifications within 1 to 10 of the terminal positions. be able to.

In some embodiments, the dsRNA molecules of the invention have 1 to 5 phosphorothioate internucleotide linkage modifications or methylphosphonate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand, and 1 to 5 phosphorothioate internucleotide linkage modifications or methylphosphonate internucleotide linkage modifications within the last 3 positions and 1 to 5 phosphorothioate nucleotides at positions 1 and 2 (counting from the 5' end) of the antisense strand It further includes an internucleotide linkage modification or a methylphosphonate internucleotide linkage modification, and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within the last six positions.

In some embodiments, the dsRNA molecules of the invention have one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate within the last six positions. internucleotide linkage modification or methylphosphonate internucleotide linkage modification, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) of the antisense strand, and the last six It further includes two phosphorothioate internucleotide linkage modifications or methylphosphonate internucleotide linkage modifications within the position.

In some embodiments, the dsRNA molecules of the invention have two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate within the last six positions. internucleotide linkage modifications, as well as one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the last six positions of the antisense strand. , further including.

In some embodiments, the dsRNA molecules of the invention have two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications within the last four positions. internucleotide linkage modifications, as well as one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the last six positions of the antisense strand. , further including.

In some embodiments, the dsRNA molecules of the invention have two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications within the last four positions. internucleotide linkage modifications, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) of the antisense strand, and one phosphorothioate internucleotide linkage modification within the last six positions. , further including.

In some embodiments, the dsRNA molecules of the invention have one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate within the last four positions. internucleotide linkage modifications, as well as two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the last six positions of the antisense strand. , further including.

In some embodiments, the dsRNA molecules of the invention have one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) of the sense strand, and one within the last six positions; and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and one phosphorothioate internucleotide linkage modification within the last six positions.

In some embodiments, the dsRNA molecules of the invention include one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) of the sense strand and ) further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and one phosphorothioate internucleotide linkage modification within the last six positions.

In some embodiments, the dsRNA molecules of the invention include two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and ) further comprises one phosphorothioate internucleotide linkage modification at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within the last six positions.

In some embodiments, the dsRNA molecules of the invention have two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and one within the last six positions; and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand, and one phosphorothioate internucleotide linkage modification within the last six positions.

In some embodiments, the dsRNA molecules of the invention have two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate within the last six positions. internucleotide linkage modifications, as well as two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the last six positions of the antisense strand. , further including.

In some embodiments, the dsRNA molecules of the invention have two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and one phosphorothioate within the last six positions. internucleotide linkage modifications, as well as one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the last six positions of the antisense strand. , further including.

In some embodiments, the dsRNA molecules of the invention have two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkages at positions 20 and 21 of the sense strand. and one phosphorothioate internucleotide linkage modification at position 1 and one at position 21 (counting from the 5' end) of the antisense strand.

In some embodiments, the dsRNA molecules of the invention have one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and It further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the antisense strand.

In some embodiments, the dsRNA molecules of the invention have two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkages at positions 21 and 22 of the sense strand. and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

In some embodiments, the dsRNA molecules of the invention have one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and It further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the antisense strand.

In some embodiments, the dsRNA molecules of the invention have two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkages at positions 22 and 23 of the sense strand. and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

In some embodiments, the dsRNA molecules of the invention have one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and It further comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 (counting from the 5' end) of the antisense strand.

In some embodiments, the sense strand includes at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 5' end of the sense strand. For example, the sense strand contains phosphorothioate linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, counting from the 5' end of the sense strand.

In some embodiments, the antisense strand includes at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 5' end of the antisense strand. For example, the antisense strand contains phosphorothioate linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, counting from the 5' end of the antisense strand.

In some embodiments, the antisense strand includes at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 3' end of the antisense strand. For example, the antisense strand contains phosphorothioate linkages between nucleotides n and n-1 and between nucleotides n-1 and n-2, where n is the length of the antisense strand, i.e. is the number of nucleotides. In other words, the antisense strand contains phosphorothioate linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, counting from the 3' end of the antisense strand.

In some embodiments, the antisense strand comprises at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 5' end of the antisense strand; contains at least two phosphorothioate internucleotide linkages between the five nucleotides of the nucleotide. For example, the antisense strand may contain between nucleotides 1 and 2 and between nucleotides 2 and 3, counting from the 5' end of the antisense strand, and between nucleotides 1 and 2, counting from the 3' end of the antisense strand, and between nucleotides 1 and 2, counting from the 3' end of the antisense strand. Contains a phosphorothioate linkage between 2 and 3.

In some embodiments, the sense strand comprises at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 5' end of the sense strand, and the antisense strand comprises at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 5' end of the antisense strand. Contains at least two phosphorothioate internucleotide linkages within the first five nucleotides. For example, the sense strand contains phosphorothioate linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, counting from the 5' end of the sense strand, and the antisense strand contains phosphorothioate linkages, counting from the 5' end of the antisense strand. Contains a phosphorothioate linkage between 1 and 2 and between nucleotides 2 and 3.

In some embodiments, the sense strand comprises at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 5' end of the sense strand, and the antisense strand comprises at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 3' end of the antisense strand. Contains at least two phosphorothioate internucleotide linkages within the first five nucleotides. For example, the sense strand contains phosphorothioate linkages between nucleotides 1 and 2 and between nucleotides 2 and 3, counting from the 5' end of the sense strand, and the antisense strand contains phosphorothioate linkages, counting from the 3' end of the antisense strand. Contains a phosphorothioate linkage between 1 and 2 and between nucleotides 2 and 3.

In some embodiments, compounds of the invention contain a pattern of backbone chiral centers. In some embodiments, the common pattern of backbone chiral centers comprises at least five internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least six internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least seven internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least eight internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least nine internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the consensus pattern of backbone chiral centers contains eight or fewer internucleotide linkages in the Rp configuration. In some embodiments, the consensus pattern of backbone chiral centers contains no more than seven internucleotide linkages in the Rp configuration. In some embodiments, the consensus pattern of backbone chiral centers contains no more than 6 internucleotide linkages in the Rp configuration. In some embodiments, the consensus pattern of backbone chiral centers includes five or fewer internucleotide linkages in the Rp configuration. In some embodiments, the consensus pattern of backbone chiral centers contains no more than four internucleotide linkages of the Rp configuration. In some embodiments, the consensus pattern of backbone chiral centers contains no more than three internucleotide linkages of the Rp configuration. In some embodiments, the consensus pattern of backbone chiral centers includes no more than two internucleotide linkages in the Rp configuration. In some embodiments, the consensus pattern of backbone chiral centers includes no more than one internucleotide linkage of the Rp configuration. In some embodiments, the consensus pattern of backbone chiral centers contains eight or fewer non-chiral internucleotide linkages (as a non-limiting example, phosphodiester). In some embodiments, the common pattern of backbone chiral centers contains seven or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers contains no more than 6 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers contains five or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers contains four or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers contains three or fewer non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers contains no more than two non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers includes one or less non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and no more than 8 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and no more than 7 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and no more than 6 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 non-chiral internucleotide linkages. In some embodiments, internucleotide linkages in the Sp configuration can be optionally contiguous or noncontiguous. In some embodiments, the internucleotide linkages of the Rp configuration may be optionally contiguous or noncontiguous. In some embodiments, non-chiral internucleotide linkages can be optionally contiguous or non-contiguous.

In some embodiments, compounds of the invention include blocks that are stereochemical blocks. In some embodiments, the block is an Rp block where each internucleotide junction of the block is an Rp. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, the block is an Sp block where each internucleotide linkage of the block is Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, provided oligonucleotides include both Rp and Sp blocks. In some embodiments, provided oligonucleotides include one or more Rp blocks but no Sp blocks. In some embodiments, provided oligonucleotides include one or more Sp blocks but no Rp blocks. In some embodiments, provided oligonucleotides include one or more PO blocks where each internucleotide linkage is a natural phosphate linkage.

In some embodiments, compounds of the invention contain a 5'-block where each sugar moiety is an Sp block containing a 2'-fluoro modification. In some embodiments, the 5'-block is an Sp block where each of the internucleotide linkages is a modified internucleotide linkage and each sugar moiety includes a 2'-fluoro modification. In some embodiments, the 5'-block is an Sp block in which each of the internucleotide linkages is a phosphorothioate linkage and each sugar moiety includes a 2'-fluoro modification. In some embodiments, the 5'-block contains four or more nucleoside units. In some embodiments, the 5'-block includes 5 or more nucleoside units. In some embodiments, the 5'-block contains 6 or more nucleoside units. In some embodiments, the 5'-block contains 7 or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety includes a 2'-fluoro modification. In some embodiments, the 3'-block is an Sp block where each of the internucleotide linkages is a modified internucleotide linkage and each sugar moiety includes a 2'-fluoro modification. In some embodiments, the 3'-block is an Sp block where each of the internucleotide linkages is a phosphorothioate linkage and each sugar moiety includes a 2'-fluoro modification. In some embodiments, the 3'-block includes four or more nucleoside units. In some embodiments, the 3'-block includes 5 or more nucleoside units. In some embodiments, the 3'-block contains 6 or more nucleoside units. In some embodiments, the 3'-block contains 7 or more nucleoside units.

In some embodiments, the compounds of the invention include a region or type of nucleoside in an oligonucleotide that is followed by a particular type of internucleotide linkage, e.g., a natural phosphate linkage, a modified internucleotide linkage. , Rp chiral internucleotide linkage, Sp chiral internucleotide linkage, and the like. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, the U is followed by a natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, the C and U are followed by a natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

Various publications have described multimeric siRNAs, any of which can be used in the dsRNAs of the present invention. Such publications include WO2007/091269, US Patent No. 7858769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520, which are incorporated herein in their entirety.

Ligands A wide variety of entities can be coupled to the dsRNA agents described herein. A preferred moiety is a ligand, which is preferably coupled covalently, either directly or indirectly through an intervening tether. Generally, the ligand alters the distribution, targeting, or lifetime of the dsRNA described herein into which it is incorporated. In some embodiments, the ligand is associated with a selected target, e.g., a molecule, cell or cell type, compartment, receptor, e.g. compartment of a cell or organ, tissue, organ or region of the body, e.g. lacking such a ligand. confers enhanced affinity compared to molecular species. Ligands that provide enhanced affinity for a selected target are also referred to herein as targeting ligands.

Some ligands can have endosomolytic properties. Endosomolytic ligands promote endosomal lysis and/or transport of a composition of the invention or a component thereof from the endosome to the cytoplasm of the cell. Endosomolytic ligands can be polyanionic peptides or peptidomimetics that exhibit pH-dependent membrane activity and fusogenic properties. In some embodiments, the endosomolytic ligand assumes its active conformation at endosomal pH. The "active" conformation is the conformation in which the endosomolytic ligand promotes endosomal lysis and/or transport of the composition of the invention or its components from the endosome to the cytoplasm of the cell. Exemplary endosomolytic ligands include the GALA peptide (Subbarao et al., Biochemistry, 1987, 26:2964-2972, herein incorporated by reference in its entirety), the EALA peptide (Vogel et al. , J. Am. Chem. Soc., 1996, 118:1581-1586, which is incorporated herein by reference in its entirety), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68, which is incorporated herein by reference in its entirety. In some embodiments, endosomolytic components may contain chemical groups (eg, amino acids) that will undergo a change in charge or protonation in response to changes in pH. The endosomolytic component can be linear or branched.

The ligand improves the transport, hybridization, and specificity properties of the resulting natural or modified oligoribonucleotides or of polymer molecules comprising any combination of monomers and/or natural or modified ribonucleotides described herein. and nuclease resistance can also be improved.

Ligands generally may include therapeutic modifiers, such as to enhance uptake; diagnostic compounds or reporter groups, such as to monitor distribution; cross-linking agents; and nuclease resistance-conferring moieties. . Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines and peptidomimetics.

Ligands include natural substances such as proteins (e.g. human serum albumin (HSA), low density lipoproteins (LDL), high density lipoproteins (HDL), or globulins); carbohydrates (e.g. dextran, pullulan, chitin, chitosan, inulin); , cyclodextrin or hyaluronic acid); or lipids. The ligand may be a recombinant or synthetic molecule, such as a synthetic polymer, such as a synthetic polyamino acid, an oligonucleotide (eg, an aptamer). Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethyl acrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary polyamines. salts, or alpha-helical peptides.

The ligand may include a targeting group, such as a cell or tissue targeting agent, such as a lectin, a glycoprotein, a lipid or a protein, such as an antibody, a nanobody, or an antibody or nanobody, such as a kidney cell or a cell of the blood-brain barrier. Moieties that bind to other cell types may also be included. Targeting groups include thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, are glycosylated polyamino acids, polyvalent galactose, transferrin targeting groups, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, biotin, RGD peptides, RGD peptidomimetics or aptamers be able to.

Other examples of ligands include dyes, intercalators (e.g. acridines), crosslinkers (e.g. psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphirin), polycyclic aromatic hydrocarbons (e.g. phenazine, dihydrophenazine). , artificial endonucleases or chelating agents (e.g. EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl groups, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and Peptide conjugates (e.g. Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino, mercapto, PEG (e.g. PEG-40K), MPEG, [MPEG] ₂ , polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes , haptens (e.g. biotin), transport/absorption enhancers (e.g. aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g. imidazoles, bisimidazoles, histamines, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles). , dinitrophenyl, HRP or AP.

The ligand may be a protein, e.g. a glycoprotein, or a peptide, e.g. a molecule with specific affinity for the co-ligand, or an antibody, e.g. an antibody that binds to a specified cell type, such as a cancer cell, an endothelial cell or a bone cell. can be. Ligands can also include hormones and hormone receptors. They also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polylactose, polygalactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polymannose, polyfucose, or aptamers. can be included. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

A ligand is a substance that can increase the uptake of an iRNA agent into a cell, e.g. by disrupting the cytoskeleton of that cell, e.g. by disrupting the microtubules, microfibrils and/or intermediate filaments of the cell. , for example a drug. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

Ligands can increase dsRNA uptake into cells, eg, by activating an inflammatory response. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, or gamma interferon.

In some embodiments, the ligand is a lipid or lipid-based molecule. Such lipids or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). The HSA-binding ligand enables distribution of the conjugate to target tissues, such as non-kidney target tissues of the body. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules capable of binding HSA can also be used as ligands. For example naproxen or aspirin can be used. The lipid or lipid-based ligand can (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to a target cell or cell membrane, and/or (c) Can be used to modulate binding to serum proteins such as HSA. Lipid-based ligands can be used to modulate, eg, control, binding of the conjugate to target tissues. For example, the more strongly a lipid or lipid-based ligand binds to HSA, the less likely it will be targeted to the kidneys and therefore the less likely it will be cleared from the body. Lipids or lipid-based ligands that bind weakly to HSA can be used to target the conjugate to the kidney.

In one preferred embodiment, the lipid-based ligand binds HSA. Preferably, it binds HSA with sufficient affinity such that the conjugate will be favorably distributed to non-kidney tissues. However, the affinity is preferably not so strong that HSA-ligand binding is no longer reversible.

In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate will be favorably distributed to the kidneys. Other moieties that target kidney cells can also be used instead of, or in addition to, lipid-based ligands.

In some embodiments, the ligand is a moiety, eg, a vitamin, that is taken up by the target cell, eg, a proliferating cell. They are particularly useful for treating disorders characterized by unnecessary cell proliferation, eg, of malignant or non-malignant types, eg, cancer cells. Exemplary vitamins include vitamins A, E and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HAS, low density lipoproteins (LDL) and high density lipoproteins (HDL).

In another aspect, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the agent is amphiphilic. Exemplary agents are peptides such as tat or antennapedia. If the agent is a peptide, it can be modified including the use of peptidyl mimetics, invertomers, non-peptide or pseudopeptide linkages, and D-amino acids. The helical agent is preferably an alpha-helical agent, preferably one having a lipophilic phase and a lipophobic phase.

を有するRFGFである。疎水性MTSを含有するRFGF類似体（例えばアミノ酸配列

）も標的化部分であることができる。ペプチド部分は、ペプチド、オリゴヌクレオチドおよびタンパク質を含む大きな極性分子を細胞膜越しに運ぶ「送達」ペプチドであることができる。例えばHIV Tatタンパク質由来の配列:

およびショウジョウバエ（Drosophila）アンテナペディアタンパク質由来の配列:

は、送達ペプチドとして機能できることがわかっている。ペプチドまたはペプチドミメティックは、ファージディスプレイライブラリーまたは1ビーズ1化合物（one-bead-one-compound:OBOC）コンビナトリアルライブラリーから同定されるペプチドなど、DNAのランダム配列によってコードされうる（Lam et al.,Nature,354:82-94,1991、この文献は参照によりその全体が本明細書に組み入れられる）。組み込まれているモノマー単位を介してiRNA剤に繋がれるペプチドまたはペプチドミメティックは、好ましくは、アルギニン-グリシン-アスパラギン酸（RGD）ペプチドまたはRGDミミックなどの細胞標的化ペプチドである。ペプチド部分の長さは約5アミノ酸～約40アミノ酸の範囲にあることができる。ペプチド部分は、例えば安定性を増加させ、またはコンフォメーション特性を方向付けるなどのために、構造修飾を有することができる。後述する構造修飾はいずれも利用可能である。RGDペプチド部分は、内皮腫瘍細胞または乳がん腫瘍細胞などの腫瘍細胞を標的とするために使用することができる（Zitzmann et al.,Cancer Res.,62:5139-43,2002、この文献は参照によりその全体が本明細書に組み入れられる）。RGDペプチドは、肺、腎臓、脾臓または肝臓を含むさまざまな他の組織の腫瘍へのiRNA剤の標的化を容易にすることができる（Aoki et al.,Cancer Gene Therapy 8:783-787,2001、この文献は参照によりその全体が本明細書に組み入れられる）。好ましくは、RGDペプチドは、腎臓へのiRNA剤の標的化を容易にするだろう。RGDペプチドは線状または環状であることができ、特異的組織への標的化を容易にするために、修飾、例えばグリコシル化、またはメチル化されていてもよい。例えばグリコシル化RGDペプチドは、iRNA剤を、α_Vβ₃を発現する腫瘍細胞に送達することができる（Haubner et al.,Jour.Nucl.Med.,42:326-336,2001、この文献は参照によりその全体が本明細書に組み入れられる）。増殖細胞において濃縮されるマーカーを標的とするペプチドを使用することができる。例えば、RGD含有ペプチドおよびペプチドミメティックは、がん細胞、特にインテグリンを呈する細胞を標的とすることができる。したがってRGDペプチド、RGDを含有する環状ペプチド、D-アミノ酸を含むRGDペプチド、ならびに合成RGDミメティックを使用することができるだろう。RGDに加えて、インテグリンリガンドを標的とする他の部分も使用することができる。一般に、そのようなリガンドは、増殖細胞および血管新生を制御するために使用することができる。このタイプの好ましいコンジュゲートは、PECAM-1、VEGFまたは他のがん遺伝子、例えば本明細書記載のがん遺伝子を標的とするリガンドを有する。 The ligand can be a peptide or a peptidomimetic. Peptidomimetics (also referred to herein as oligopeptide mimetics) are molecules that can fold into well-defined three-dimensional structures similar to natural peptides. A peptide or peptidomimetic moiety can be about 5-50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in length. The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphiphilic peptide or a hydrophobic peptide (eg, one consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide or a cross-linked peptide. As another option, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). Exemplary hydrophobic MTS-containing peptides have amino acid sequences

RFGF with RFGF analogs containing a hydrophobic MTS (e.g. amino acid sequence

) can also be a targeting moiety. Peptide moieties can be "delivery" peptides that carry large polar molecules, including peptides, oligonucleotides and proteins, across cell membranes. For example, sequences from the HIV Tat protein:

and sequences from Drosophila Antennapedia protein:

have been shown to be able to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al. , Nature, 354:82-94, 1991, which is incorporated herein by reference in its entirety). The peptide or peptidomimetic linked to the iRNA agent via the incorporated monomer unit is preferably a cell-targeting peptide, such as an arginine-glycine-aspartate (RGD) peptide or an RGD mimic. The length of the peptide portion can range from about 5 amino acids to about 40 amino acids. Peptide moieties can have structural modifications, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be used. RGD peptide moieties can be used to target tumor cells such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002, this article is incorporated by reference. (incorporated herein in its entirety). RGD peptides can facilitate targeting of iRNA agents to tumors in a variety of other tissues including lung, kidney, spleen or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001 , this document is incorporated herein by reference in its entirety). Preferably, the RGD peptide will facilitate targeting of the iRNA agent to the kidney. RGD peptides can be linear or cyclic and may be modified, such as glycosylated or methylated, to facilitate targeting to specific tissues. For example, glycosylated RGD peptides can deliver iRNA agents to tumor cells expressing α _V β ₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001, this article (incorporated herein by reference in its entirety). Peptides that target markers that are enriched in proliferating cells can be used. For example, RGD-containing peptides and peptidomimetics can target cancer cells, particularly cells that exhibit integrins. Thus, RGD peptides, cyclic peptides containing RGD, RGD peptides containing D-amino acids, as well as synthetic RGD mimetics could be used. In addition to RGD, other moieties that target integrin ligands can also be used. Generally, such ligands can be used to control cell proliferation and angiogenesis. Preferred conjugates of this type have ligands that target PECAM-1, VEGF or other oncogenes, such as those described herein.

A "cell-penetrating peptide" is capable of penetrating a cell, eg, a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g. LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g. α-defensins, β-defensins or bactenecins), or only one or It can be a peptide containing predominantly two amino acids (eg PR-39 or indolicidin). Cell penetrating peptides can also include nuclear localization signals (NLS). For example, the cell penetrating peptide can be a bipartite amphipathic peptide derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen, such as MPG (Simeoni et al., Nucl. Acids Res .31:2717-2724, 2003, which is incorporated herein by reference in its entirety).

In some embodiments, the targeting peptide can be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antiseptic. These include microbial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, _H2A peptide, Xenopus peptide, esculentinis-1, and caerin. , but not limited to. In order to maintain the integrity of helical stability, several factors will preferably be considered. For example, the maximum number of helix stabilizing residues will be utilized (eg leu, ala or lys) and the minimum number of helix destabilizing residues (eg proline or cyclic monomer units). Capping residues may be considered (e.g. Gly is an exemplary N-capping residue, and/or C-terminal amidation is used to provide extra H-bonds to stabilize the helix). The formation of salt bridges between oppositely charged residues i±3 or i±4 apart can provide stability. For example, lysine, arginine, homo-arginine, ornithine or histidine, etc. The cationic residues of can form salt bridges with anionic residues such as glutamic acid or aspartic acid.

Peptides and peptidomimetic ligands include naturally occurring or modified peptides, such as D or L peptides; α, β or γ peptides; N-methyl peptides; aza peptides; one or more amide linkages, i.e. peptide linkages. , peptides that are replaced with one or more urea, thiourea, carbamate, or sulfonylurea linkages; or those with cyclic peptides.

A targeting ligand can be any ligand capable of targeting a specific receptor. Examples are folic acid, GalNAc, galactose, mannose, mannose-6P, clusters of sugars such as GalNAc clusters, mannose clusters, galactose clusters, or aptamers. A cluster is a combination of two or more sugar units. Targeting ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. Ligands can also be based on nucleic acids, such as aptamers. Aptamers can be unmodified or have any combination of modifications disclosed herein.

As endosomal release agents, imidazoles, poly or oligoimidazole, PEI, peptides, fusogenic peptides, polycarboxylates, polycations, shielding oligos or polycations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, Included are polymers with shielded or unshielded cations or anionic charges, dendrimers with shielded or unshielded cations or anionic charges.

PK modulator means a pharmacokinetic modulator. PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. isn't it. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins, and therefore short oligonucleotides containing multiple phosphorothioate linkages in the backbone, such as about 5 bases, 10 bases, 15 bases or 20 bases, are also known to bind to serum proteins. Base oligonucleotides are also applicable to the present invention as ligands (eg, as PK modulating ligands).

In addition, aptamers that bind serum components (eg serum proteins) are also applicable to the present invention as PK modulating ligands.

Other ligand conjugates applicable to the present invention are described in the following U.S. patent applications, each of which is incorporated herein by reference in its entirety for all purposes: U.S. Patent Application No. 10/916,185, filed September 21, 2004, U.S. Patent Application No. 10/946,873, filed August 3, 2007, U.S. Patent Application No. 10/833,934, 2005 U.S. Patent Application No. 11/115,989, filed April 27, and U.S. Patent Application No. 11/944,227, filed November 21, 2007.

In some embodiments, a dsRNA molecule can include more than one ligand, such as 2, 3, 4 or 5 ligands. If two or more ligands are present, they may all have the same properties, all have different properties, or some ligands may have the same properties while others have different properties. can have For example, a ligand can have targeting properties, endosomolytic activity, or PK modulating properties. In one preferred embodiment, all the ligands have different properties.

In some embodiments, the dsRNA molecule includes two ligands. For example, the sense strand of a dsRNA molecule includes a first ligand attached to the 3' end of the sense strand and a second ligand attached to the 5' end of the sense strand. In some embodiments, the dsRNA molecule includes two ligands linked to the sense strand, the first ligand containing an inverted abasic nucleotide (i.e., an abasic nucleotide linked by a 3'→3' junction). and the second ligand includes an ASGPR ligand.

Ligands can be coupled to dsRNA at various locations, such as at the 3' end, 5' end, and/or internal positions of the sense and/or antisense strands. In a preferred embodiment, the ligand is attached to the sense and/or antisense strand of the dsRNA via a linker or tether. A ligand or tethered ligand can be present on the monomer as it is incorporated into the growing chain. In some embodiments, the ligand can be incorporated into the "precursor" monomer after it has been incorporated into the growing chain. For example, a monomer with an amino-terminated tether (ie, without _a ligand), such as TAP-( _CH2 ) _nNH2 , can be incorporated into a growing oligonucleotide chain. In a subsequent operation, i.e. after incorporation of the precursor monomer into the chain, a ligand with an electrophilic group, such as a pentafluorophenyl ester or an aldehyde group, is subsequently attached to the end of the tether of the electrophilic group of the ligand and the precursor monomer. It can be attached to its precursor monomer by coupling with a nuclear group.

In another example, monomers with suitable chemical groups to participate in click chemistry reactions, such as, for example, azide or alkyne-terminated tethers/linkers, may be incorporated. In a subsequent operation, i.e. after incorporation of the precursor monomer into the chain, a ligand with a complementary chemical group, e.g. an alkyne or an azide, is attached to the precursor monomer by coupling the alkyne and the azide together. I can do it.

Ligands can be attached to one or both strands. In some embodiments, the dsRNA described herein includes a ligand conjugated to the sense strand. In some embodiments, the dsRNA described herein includes a ligand conjugated to an antisense strand.

In some embodiments, the ligand is conjugated to the sense strand. As described herein, the ligand can be conjugated at the 3' end, 5' end, or an internal position of the sense strand. In some embodiments, the ligand is conjugated to the 3' end of the sense strand. In some embodiments, the ligand is conjugated to the 5' end of the sense strand. In some embodiments, the ligand is conjugated at an internal position on the sense strand. In other words, the ligand is conjugated to a non-terminal nucleotide of the sense strand. Note that the ligand can be conjugated to the nucleobase, sugar moiety or internucleotide linkage of the sense strand.

In some embodiments, the ligand is conjugated at the 2' position of the nucleotide in the sense strand. For example, the ligand is conjugated at the 2' position of a nucleotide in an internal position, ie, a non-terminal position, of the sense strand.

In some embodiments, a ligand can be conjugated to a nucleobase, sugar moiety, or internucleoside linkage of a nucleic acid molecule. Conjugation to a purine nucleobase or derivative thereof can be made at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7- or 8-position of the purine nucleobase is attached to the conjugate moiety. Conjugation to a pyrimidine nucleobase or derivative thereof can also be performed at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase can be replaced with conjugate moieties. Conjugation of the nucleoside to the sugar moiety can occur at any carbon atom. Examples of sugar moiety carbon atoms that can be attached to the conjugate moiety include 2', 3' and 5' carbon atoms. The 1' position can also be attached to the conjugate, such as with abasic residues. Internucleoside linkages can also carry conjugate moieties. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the conjugate moiety can be attached directly to the phosphorus atom or the O, N or S Can be attached to atoms. For internucleoside linkages containing amines or amides (eg, PNA), the conjugate moiety can be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

In some embodiments, the ligand is conjugated to the sense strand. As described herein, the ligand can be conjugated at the 3' end, 5' end, or an internal position of the sense strand. In some embodiments, the ligand is conjugated to the 3' end of the sense strand. Additionally, it is noted that the ligand can be conjugated to the nucleobase, sugar moiety or internucleotide linkage of the sense strand.

Although any suitable ligand in the field of RNA interference may be used, the ligand is typically a carbohydrate, such as a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, a polysaccharide.

Linkers for conjugating a ligand to a nucleic acid include those described above. For example, the ligand can be a GalNAc (N-acetylgalactosamine) derivative attached by one or more carbohydrates, such as monovalent, divalent or trivalent branched linkers.

式中、
q^2A、q^2B、q^3A、q^3B、q4^A、q^4B、q^5A、q^5Bおよびq^5Cは、出現ごとに独立して、0～20を表し、繰り返し単位は同じであるか、または異なることができ、
P^2A、P^2B、P^3A、P^3B、P^4A、P^4B、P^5A、P^5B、P^5C、T^2A、T^2B、T^3A、T^3B、T^4A、T^4B、T^5A、T^5B、T^5Cはそれぞれ、出現ごとに独立して、存在しないか、またはCO、NH、O、S、OC（O）、NHC（O）、CH₂、CH₂NHもしくはCH₂Oであり、
Q^2A、Q^2B、Q^3A、Q^3B、Q^4A、Q^4B、Q^5A、Q^5B、Q^5Cは、出現ごとに独立して、存在しないか、またはアルキレン、置換アルキレンであり、ここで、1つまたは複数のメチレンは、O、S、S（O）、SO₂、N（R^N）、C（R'）＝C（R”）、C≡CまたはC（O）のうちの1つまたは複数で中断されるか終わっていてもよく、
R^2A、R^2B、R^3A、R^3B、R^4A、R^4B、R^5A、R^5B、R^5Cはそれぞれ、出現ごとに独立して、存在しないか、またはNH、O、S、CH₂、C（O）O、C（O）NH、NHCH（R^a）C（O）、-C（O）-CH（R^a）-NH-、CO、CH＝N-O、

もしくはヘテロシクリルであり、
L^2A、L^2B、L^3A、L^3B、L^4A、L^4B、L^5A、L^5BおよびL^5Cは、リガンド、すなわち、それぞれ出現ごとに独立して、単糖（GalNAcなど）、二糖、三糖、四糖、オリゴ糖または多糖を表し、
R^aは、Hまたはアミノ酸側鎖である。 In some embodiments, the dsRNA of the invention is conjugated to divalent and trivalent branched linkers comprising the structures shown in any of Formulas (IV)-(VII):

During the ceremony,
q ^2A , q ^2B , q ^3A , q ^3B , q4 ^A , q ^4B , q ^5A , q ^5B and q ^5C each independently represent 0 to 20, the repeating units are the same, or can be different,
P ^2A , P ^2B , P ^3A , P ^3B , P ^4A , P ^4B , P ^5A , P ^5B , P ^5C , T 2A , T ^2B , T ^3A , T ^3B , T ^4A , T ^4B , T ^5A , ^{T 5B} , T ^5C are each, independently of each occurrence, absent or CO, NH, O, S, OC(O), NHC(O), CH ₂ , CH ₂ NH or CH ₂ O;
Q ^2A , Q ^2B , Q ^3A , Q ^3B , Q ^4A , Q ^4B , Q ^5A , Q ^5B , Q ^5C are independently at each occurrence absent or alkylene, substituted alkylene, where One or more methylenes are one of O, S, S(O), SO ₂ , N(R ^N ), C(R')=C(R”), C≡C or C(O) may be interrupted or terminated by one or more;
R ^2A , R ^2B , R ^3A , R ^3B , R ^4A , R ^4B , R ^5A , R ^5B , R ^5C are each independently at each occurrence either absent or NH, O, S, CH ₂ , C(O)O, C(O)NH, NHCH(R ^a )C(O), -C(O)-CH(R ^a )-NH-, CO, CH=NO,

or heterocyclyl;
L ^2A , L ^2B , L ^3A , L ^3B , L ^4A , L ^4B , L ^5A , L ^5B and L ^5C are ligands, i.e., monosaccharides (such as GalNAc), disaccharides, represents a trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide;
R ^a is H or an amino acid side chain.

のものなど、GalNAc誘導体をコンジュゲートする三価は、標的遺伝子の発現を阻害するための本明細書記載のdsRNA剤に使用するのに、とりわけ有用であり、
式中、L^5A、L^5BおよびL^5Cは、GalNAc誘導体などの単糖である。 For example, formula (VII):

Trivalent conjugates of GalNAc derivatives, such as those described herein, are particularly useful for use in the dsRNA agents described herein to inhibit expression of target genes;
where L ^5A , L ^5B and L ^5C are monosaccharides such as GalNAc derivatives.

。 Suitable divalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the following compounds:

.

。 In some embodiments, the dsRNA described herein comprises Ligand 1, a ligand having the following structure:

.

In some embodiments, the dsRNA described herein comprises a ligand described in US Pat. No. 5,994,517 or US Pat. No. 6,906,182, the contents of each of which are incorporated herein by reference in their entirety.

。 In some embodiments, the ligand can be a triantennary ligand as described in FIG. 3 of US Pat. No. 6,906,182. For example, the dsRNA described herein can include a ligand selected from the following triantennary ligands:

.

In some embodiments of any one of the above aspects, the antisense strand includes a phosphoryl analog or phosphate mimic at the 5' end. In some embodiments, the antisense strand includes an alkenylphosphonate, ie, a vinylphosphonate, at the 5' end. For example, the antisense strand includes 5'-E-vinylphosphonate.

を含み、式中、＊は、5'末端にあるヌクレオチドのC5位への結合である。 In some embodiments, the antisense strand includes a cyclopropylphosphonate at the 5' end. For example, antisense is

where * is the attachment of the nucleotide at the 5' end to the C5 position.

式中、＊は鎖の5',3'末端ヒドロキシル基への結合である;

式中、＊は鎖中のヌクレオチドの2'-ヒドロキシルへの結合である;

式中、＊は、鎖の5'末端または3'末端への結合である;

式中、＊は、鎖の5'末端または3'末端への結合である;

式中、-O-＊は、鎖の5'末端または3'末端への接続である;および/または

式中、-P-＊は、鎖の5'または3'-ヒドロキシル基への接続である。 In some embodiments of any one of the above aspects, at least one of the strands, e.g., the sense and/or antisense strand of the double-stranded RNA, comprises a monomer or ligand selected from:

where * is the bond to the 5',3' terminal hydroxyl group of the chain;

where * is the bond to the 2'-hydroxyl of the nucleotide in the chain;

where * is a bond to the 5' or 3' end of the chain;

where * is a bond to the 5' or 3' end of the chain;

where -O-* is a connection to the 5' or 3' end of the chain; and/or

where -P-* is the attachment to the 5' or 3'-hydroxyl group of the chain.

In some embodiments, the ligand includes a lipophilic group. For example, the ligand can be a C _6-30 aliphatic group or a C _10-30 aliphatic group. "Aliphatic" as used herein means a saturated or unsaturated straight chain, branched and/or cyclic hydrocarbon having the specified number of carbon atoms, e.g. Mention may be made of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylakenyl and cycloalkylalkynyl having carbon atoms. In some embodiments, the ligand is a C _10-30 alkyl group. For example, the ligand is a straight chain or branched hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, or tetracosyl group. For example, the ligand is a straight chain hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, or tetracosyl group. For example, the ligand is a straight chain hexyl, octyl, decyl, dodecyl, hexadecyl, octadecyl, icosyl, or docosyl group. For example, the ligand is a straight chain hexadecyl group. For example, the ligand is a straight chain docosyl group.

In certain embodiments, the ligand is conjugated at the 2' position of the nucleotide in an internal, non-terminal position of the sense strand and is a linear or branched tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, or tetracosyl group. For example, the ligand may be conjugated within the sense strand, i.e. at the 2' position of a nucleotide in a non-terminal position, with a linear or branched hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, or tetracosyl group. It is. For example, the ligand is conjugated at the 2' position of the nucleotide in the interior of the sense strand, i.e. in a non-terminal position, and is a linear hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, or tetracosyl group. . For example, the ligand is conjugated at the 2' position of the nucleotide in an internal, non-terminal position of the sense strand and is a linear hexadecyl, octadecyl, icosyl, or docosyl group.

を有し、式中、Bはヌクレオチド塩基またはヌクレオチド塩基類似体であり、任意で、Bはアデニン、グアニン、シトシン、チミンまたはウラシルである。 Internal sense strand nucleotide positions can be at any position except the three terminal positions from each end of the sense strand. In some embodiments, the internal location excludes the sense strand cleavage site region. In some embodiments, the internal positions exclude positions 9-12 or positions 11-13 counting from the 5' end of the sense strand. For example, internal nucleotide positions may include one or more of positions 4-8 and 13-18 on the sense strand, counting from the 5' end of the sense strand, such as positions 5, 6, 7, 15 on the sense strand. and one or more of 17. In one embodiment, the internal nucleotide position can be one of positions 5, 6, 7 or 8 of the sense strand counting from the 5' end. For example, in each of these embodiments, the internal nucleotide position is position 6 or 7 of the sense strand counting from the 5' end. For example, in each of these embodiments, the internal nucleotide position is position 6 of the sense strand, counting from the 5' end. For example, in each of these embodiments, the internal nucleotide position is position 7 of the sense strand, counting from the 5' end. In certain embodiments, the internal nucleotide comprising the ligand has the formula:

where B is a nucleotide base or nucleotide base analog, and optionally B is adenine, guanine, cytosine, thymine or uracil.

In some embodiments, the ligand comprises an inverted nucleotide or an inverted abasic nucleotide. For example, the ligand includes an abasic nucleotide linked to the strand of the dsRNA molecule via a 5'→5' or 3'→3' linkage. In some embodiments, the ligand comprises an abasic nucleotide linked to the 3' end of the sense strand via a 3'→3' linkage.

が含まれる。例えば、そのようなリガンドは、dsRNA分子の鎖の5'および/または3'端に取り付けられうる。いくつかの態様において、リガンドはセンス鎖の5'または3'端に取り付けられうる。いくつかの態様において、リガンドはセンス鎖の5'端に取り付けられうる。いくつかの態様において、リガンドはセンス鎖の3'端に取り付けられうる。dsRNA分子への取付けは、空の原子価を有する、上に図解した酸素原子への結合、またはピロリジンの4-ヒドロキシル基を使って形成される結合によって行いうる。 In another embodiment, the ligand includes a lipophilic group that includes a fused ring system of a steroid. For example, the ligand can include cholesterol or corticosterone. Examples of such ligands include, e.g.

is included. For example, such a ligand can be attached to the 5' and/or 3' end of a strand of a dsRNA molecule. In some embodiments, the ligand can be attached to the 5' or 3' end of the sense strand. In some embodiments, the ligand can be attached to the 5' end of the sense strand. In some embodiments, the ligand can be attached to the 3' end of the sense strand. Attachment to the dsRNA molecule can be by a bond to an oxygen atom, as illustrated above, with an empty valence, or by a bond formed using the 4-hydroxyl group of pyrrolidine.

A ligand may be attached to a polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point", preferably two "backbone attachment points", and (ii) at least one "tethering attachment point". A "backbone attachment point" as used herein refers to a functional group that is available and suitable for incorporation of a carrier into the backbone of a ribonucleic acid, such as a phosphate backbone, or a modified phosphate, such as a sulfur-containing backbone. For example, it refers to a hydroxyl group, or broadly to a bond. A "tethering attachment point" (TAP), in some embodiments, refers to a constituent ring atom of a cyclic carrier, such as a carbon atom or a heteroatom (different from the atom that provides the backbone attachment point), that connects selected moieties. ). Said moiety can be, for example, a carbohydrate, such as monosaccharides, di-saccharides, trisaccharides, tetrasaccharides, oligosaccharides and polysaccharides. Optionally, selected portions are connected to the annular carrier by intervening tethers. The cyclic carrier will therefore often contain, or broadly will provide, functional groups suitable for the incorporation or tethering of another chemical entity, e.g. a ligand, to the constituent ring, e.g. an amino group. .

In one embodiment, the dsRNA molecule of the invention is conjugated to the ligand via a carrier, the carrier can be a cyclic group or an acyclic group, preferably the cyclic group is pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, selected from piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin, preferably the acyclic group is serinol backbone or diethanolamine backbone.

The ligand can be attached to the sense strand, antisense strand or both strands at the 3' end, 5' end or both ends. For example, a ligand can be conjugated to the sense strand, particularly to the 3' end of the sense strand.

The ligand can be conjugated to the sense or antisense strand via a linker containing a cleavable group. A cleavable linking group is a linking group that is sufficiently stable outside the cell, but is cleaved once inside the target cell, releasing the two moieties that the linker connects together. In a preferred embodiment of the dsRNA molecule of the invention, the cleavable linking group is cleavable within the target cell or under a first reference condition (which can be chosen to, for example, mimic or represent intracellular conditions). at least 10 times faster, preferably at least 100 times faster than in blood or under second reference conditions (which can be chosen to mimic or represent conditions found in blood or serum, for example). cut off.

Cleavable linking groups are sensitive to cleavage agents such as pH, redox potential or the presence of degradable molecules. Generally, cleavage agents are found predominantly or at higher levels or activity inside cells than in serum or blood. Examples of such degradable agents include redox agents selected for a particular substrate or redox agents without substrate specificity, such as oxidases or reductases, or reducing redox-cleavable linking groups. Reducing agents such as mercaptans present in cells that can be degraded by esterases; endosomes or agents that can create an acidic environment, e.g. those that result in a pH below 5; acid-cleavable linking groups, acting as general acids There are enzymes, peptidases (which can be substrate specific) and phosphatases, which can be hydrolyzed or degraded by

Cleavable linking groups such as disulfide bonds can be sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes are more acidic, with a pH range of 5.5 to 6.0, and lysosomes have an even more acidic pH, around 5.0. Some linkers will have a cleavable linking group that upon cleavage at a preferred pH releases the cationic lipid from the ligand within the cell or to a desired compartment of the cell.

The linker can include a cleavable linking group that is cleavable by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the targeted cell. For example, a liver targeting ligand can be linked to a cationic lipid via a linker containing an ester group. Because liver cells are rich in esterases, linkers will be cleaved more efficiently in liver cells than in cell types that are less rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

Linkers containing peptide bonds can be used when targeting peptidase-rich cell types, such as liver cells and synovial cells.

Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradable agent (or condition) to cleave the candidate linking group. It would also be desirable to test candidate cleavable linking groups for their ability to resist cleavage in blood or when contacted with other non-target tissues. Thus, a relative relationship between the first condition selected to indicate cleavage in the target cell and the second condition selected to indicate cleavage in other tissues or biological fluids, such as blood or serum, is determined. Susceptibility can be determined. Evaluations can be performed in cell-free systems, in cells, in cell cultures, in organs or tissues, or in whole animals. It may be useful to perform initial evaluations under cell-free or cell culture conditions and confirm by further evaluation in whole animals. In a preferred embodiment, useful candidate compounds are intracellular (or selected to mimic intracellular conditions) as compared to blood or serum (or in vitro conditions selected to mimic extracellular conditions). (in vitro conditions) at least 2x, 4x, 10x or 100x faster.

One class of cleavable linking groups are redox-cleavable linking groups, which can be used in dsRNA molecules of the invention that are cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-S-S-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or suitable for use with a particular iRNA moiety and a particular targeting agent, for example, One can rely on the methods described herein. For example, candidates are evaluated by incubation with cells, e.g., dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rates that would be observed in target cells. can do. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In one preferred embodiment, the candidate compound is no more than 10% cleaved in blood. In a preferred embodiment, useful candidate compounds are intracellular (or in vitro conditions selected to mimic intracellular conditions) as compared to blood (or in vitro conditions selected to mimic extracellular conditions). below), it resolves at least 2x, 4x, 10x or 100x faster. The cleavage rate of the candidate compound is determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium. be able to.

Phosphate-based cleavable linking groups that can be used in dsRNA molecules of the invention are cleaved by agents that degrade or hydrolyze phosphate groups. Examples of agents that cleave phosphate groups within cells are enzymes such as phosphatases within cells. Examples of phosphate-based linking groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O) (ORk) -O-, -O-P(O)(ORk) -S-, -S-P(O)(ORk) -S-, -O-P(S)(ORk) -S-, -S-P(S)(ORk ) -O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)- O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S-, where Rk is independently at each occurrence hydrogen, C1-C20 alkyl, C1 ~C20 haloalkyl, C6-C10 aryl, C7-C12 aralkyl. Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O -, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P (O)(H)-S-, -O-P(S)(H)-S-. A preferred embodiment is -O-P(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

Acid-cleavable linking groups that can be used in the dsRNA molecules of the invention are linking groups that are cleaved under acidic conditions. In preferred embodiments, the acid-cleavable linking group is cleavable in an acidic environment at a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less) or by an agent such as an enzyme that can act as a general acid. Ru. In cells, certain low pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C=NN-, C(O)O or -OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (alkoxy group) is an aryl, substituted alkyl or tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

Ester-based cleavable linking groups that can be used in the dsRNA molecules of the invention are cleaved intracellularly by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, alkylene groups, alkenylene groups, and esters of alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

Peptide-based cleavable linking groups that can be used in dsRNA molecules of the invention are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to give oligopeptides (eg, dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not contain amide groups (-C(O)NH-). An amide group can be formed between any alkylene, alkenylene or alkynylene. Peptide bonds are a special type of amide bond that is formed between amino acids to give peptides and proteins. Peptide-based cleavage groups are generally limited to peptide bonds (ie, amide bonds) formed between amino acids to give peptides and proteins, and do not include the entire amide functionality. The peptide-based cleavable linking group has the general formula -NHCHR ^A C(O)NHCHR ^B C(O)-, where R ^A and R ^B are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above. As used herein, "carbohydrate" refers to one or more monosaccharides having at least 6 carbon atoms (which may be linear, branched or cyclic) and an oxygen, nitrogen or sulfur atom attached to each carbon atom. a carbohydrate itself made up of units, or one or more units having at least 6 carbon atoms (which may be linear, branched or cyclic) and an oxygen, nitrogen or sulfur atom attached to each carbon atom. Refers to a compound that is a compound that has as part of its carbohydrate moiety made up of sugar units. Representative carbohydrates include sugars (monosaccharides, di-saccharides, trisaccharides and oligosaccharides containing about 4 to 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include sugars with C ₅ or higher (preferably C ₅ -C ₈ ), and disaccharides and trisaccharides contain two or three monosaccharide units (preferably C ₅ -C ₈ ). Contains sugars that have

In some embodiments, the dsRNA molecules of the invention include one or more overhang regions and/or capping groups of the dsRNA molecule at the 3' or 5' ends or both ends of the strand. Overhangs can be 1-10 nucleotides in length. For example, an overhang can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. In some embodiments, the overhang is 1 to 6 nucleotides in length, such as 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length. long, 2-3 nucleotides long, or 1-2 nucleotides long. Overhang can be the result of one strand being longer than the other, or the result of two strands of the same length being offset from each other. The overhang can form a mismatch with the target sequence, be complementary to the targeted gene sequence, or be other sequences. The first and second strands can also be joined, for example, with additional bases to form a hairpin, or with other non-basic linkers.

In some embodiments, the nucleotides in the overhang region of the dsRNA molecules of the invention are each independently modified or unmodified nucleotides, such as, but not limited to, 2'-sugar modifications, such as 2'- Fluoro, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine, 2'-O-methoxyethyladenosine, 2'-O-methoxyethyl-5-methylcytidine, GNA , SNA, hGNA, hhGNA, mGNA, TNA, h'GNA, and any combination thereof. For example, dTdT can be an overhang sequence on either end of either strand. The overhang can form a mismatch with the target mRNA, be complementary to the targeted gene sequence, or be other sequences.

The 5'-overhang or 3'-overhang of the sense strand, antisense strand or both strands of the dsRNA molecules of the invention can be phosphorylated. In some embodiments, the overhang region contains two nucleotides with a phosphorothioate between the two nucleotides, and the two nucleotides may be the same or different. In some embodiments, the overhang is present at the 3' end of the sense strand, antisense strand or both strands. In some embodiments, this 3'-overhang is present on the antisense strand. In some embodiments, this 3'-overhang is present on the sense strand.

The dsRNA molecules of the invention may contain only one overhang, which can enhance its interfering activity without affecting the overall stability of the dsRNA. For example, a single-stranded overhang can be located at the 3' end of the sense strand or alternatively at the 3' end of the antisense strand. The dsRNA can also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa.

Generally, the antisense strand of dsRNA has a nucleotide overhang at the 3' end and is blunt at the 5' end. Without wishing to be bound by theory, the blunt end of the 5' end of the asymmetric antisense strand and the 3' overhang of the antisense strand are advantageous for the loading process of the guide strand into the RISC. For example, a single overhang is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides long. In some embodiments, the dsRNA has a 2 nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

dsRNAs of the invention can include one or more modified nucleotides. For example, all nucleotides in the sense and antisense strands of a dsRNA molecule can be modified. Each nucleotide can be modified with the same or different modifications, including one or more of the unlinked phosphate oxygens and/or one or more of the linked phosphate oxygens. modification; modification of the ribose sugar component; replacement of the ribose sugar; full replacement of the phosphate moiety by a "dephospho" linker; modification or substitution of natural bases; and substitution or modification of the ribose-phosphate backbone. can.

Because nucleic acids are polymers of subunits, many of the modifications occur at repeated positions within the nucleic acid, such as modification of the base or the phosphate moiety or the unlinked O of the phosphate moiety. In some cases, the modification will be present at all of the positions of interest in the nucleic acid, but in many cases this will not be the case. For example, the modification is present only in the 3' terminal position or the 5' terminal position, or only in the central region, or only in the non-terminal region, or in the terminal region, e.g. at a position on the terminal nucleotide or It may be present only in the last 2, 3, 4, 5 or 10 nucleotides of the chain. Modifications may be present in the double-stranded region, the single-stranded region, or both. Modifications can be present only in double-stranded regions of the RNA or only in single-stranded regions of the RNA. For example, the phosphorothioate modification at the unlinked O position may be present only at one or both ends, or only in the terminal region, e.g. on the terminal nucleotide, or in the last 2, 3, 4, 5 or 10 nucleotides of the chain. present or may be present in double-stranded regions and single-stranded regions, especially at the ends. One or more 5' ends can be phosphorylated.

For example, enhancing stability, including specific bases in the overhang, or including modified nucleotides or nucleotide substitutes in a single-stranded overhang, such as a 5' overhang or a 3' overhang, or both. may be possible. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' overhang or 5' overhang may be modified, eg, with the modifications described herein. Modifications include, for example, the use of modifications at the 2' position of the ribose sugar by modifications known in the art, e.g. deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'- F) or the use of 2'-O-methyl modifications, and modifications in the phosphate group, such as phosphorothioate modifications. There is no requirement that the overhang be homologous to the target sequence.

In some embodiments, the dsRNA molecules of the invention include alternating patterns of modifications, particularly in the B1, B2, B3, B1', B2', B3', B4' regions. The term "alternating motif" or "alternating pattern" as used herein refers to a motif having one or more modifications, each modification being on alternating nucleotides of one strand. Alternating nucleotides can refer to patterns such as every other nucleotide or every second nucleotide. For example, if A, B, and C each represent a type of modification to a nucleotide, then alternating motifs are “ABABABABABAB…”, “AABBAABBAABB…”, “AABAABAABAAB…”, “AAABAAABAAAB…”, “AAABBBAAABBB…”, or “ABCABCABCABC”. …” and so on.

The types of modifications included in alternating motifs may be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, then the alternating pattern, that is, the modification on every other nucleotide may be the same, but each on the sense or antisense strand can be selected from several possible modifications within the alternating motif, such as, for example, "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

In some embodiments, a dsRNA molecule of the invention comprises a modification pattern for alternating motifs on the sense strand that is shifted relative to a modification pattern for alternating motifs on the antisense strand. The shift can be such that a modified group on a nucleotide in the sense strand corresponds to a differently modified group on a nucleotide in the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, within the duplex region, the alternating motifs in the sense strand are "ABABAB" from 5' to 3' of the strand. Alternating motifs in the antisense strand can begin with "BABABA" from 3' to 5' of the strand. As another example, within a duplex region, alternating motifs in the sense strand are separated from the 5′ to 3 ', and alternating motifs in the antisense strand can begin with 'BBAABBAA' from 3' to 5' of the strand.

5'-Modifications In some embodiments, the dsRNA molecules of the invention can be 5' phosphorylated or include a phosphoryl analog or phosphate mimic at the 5' end. 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include 5'-monophosphate ((HO) ₂ (O)PO-5');5'-diphosphate ((HO) ₂ (O)POP(HO)(O)-O-5') ;5'-triphosphate ((HO) ₂ (O)PO-(HO)(O)POP(HO)(O)-O-5');5'-guanosine cap (7-methylated or unmethylated ) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5');5'-adenosine cap (Appp) and any modified nucleotides cap structure or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5');5'-monothiophosphate(phosphorothioate;(HO) ₂ (S)PO-5');5'-monodithiophosphate (phosphorodithioate;(HO)(HS)(S)PO-5'),5'-phosphorothiolate(( HO)2(O)PS-5'); any further combinations of oxygen/sulfur substituted monophosphates, diphosphates and triphosphates (e.g. 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.) , 5'-phosphoramidates ((HO) ₂ (O)P-NH-5', (HO)( _NH2 )(O)PO-5'), 5'-alkylphosphonates (e.g. RP(OH)( O) -O-5'-, R = alkyl, e.g. methyl, ethyl, isopropyl, propyl, etc.), 5'-alkenylphosphonate (i.e. vinyl, substituted vinyl), (OH) ₂ (O)P-5'-CH _2- ), cyclopropylphosphonate, 5'-alkyl ether phosphonate (R=alkyl ether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., for example, RP(OH)(O)-O-5'-). As an example, modifications can be placed on the antisense strand of a dsRNA molecule.

式中、XはOまたはSであり、Rは水素、ヒドロキシ、フルオロ、またはC_1～20アルコキシ（例えばメトキシ）であり、R^5'は＝C（H）-P（O）（OH）₂であり、C5'炭素とR5'の間の二重結合はE配向またはZ配向（例えばE配向）であり、Bは核酸塩基または修飾核酸塩基であり、任意で、Bは、アデニン、グアニン、シトシン、チミン、またはウラシルである。 In some embodiments of any one of the above aspects, the antisense strand includes a phosphoryl analog or phosphate mimic at the 5' end. In some embodiments, the antisense strand includes an alkenylphosphonate, ie, a vinylphosphonate, at the 5' end. For example, the antisense strand includes 5'-E-vinylphosphonate. In an exemplary embodiment, the 5' terminal 5' vinylphosphonate modified nucleotide can have the following structure:

where X is O or S, R is hydrogen, hydroxy, fluoro, or C _1-20 alkoxy (e.g. methoxy), and R ^5' is =C(H)-P(O)(OH) ₂ and the double bond between the C5' carbon and R5' is in the E or Z orientation (e.g. E orientation), B is a nucleobase or modified nucleobase, and optionally B is adenine, guanine, Cytosine, thymine, or uracil.

を含む（これは、例えば、直前の構造における4'基を置き換えうる。 In some embodiments, the antisense strand includes a cyclopropylphosphonate at the 5' end. For example, antisense is

(which may, for example, replace the 4' group in the immediately preceding structure).

The dsRNA agents of the invention include a thermodestabilizing modification in the seed region of the antisense strand (i.e., positions 2-9 from the 5' end of the antisense strand) to reduce or inhibit off-target gene silencing. be able to. Without wishing to be bound by theory, dsRNAs with antisense strands containing at least one duplex heat-destabilizing modification within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced It has off-target gene silencing activity. Thus, in some embodiments, the antisense strand has at least one (e.g., one, two) duplex heat-destabilizing modifications within the first nine nucleotide positions of the 5' region of the antisense strand. , 3, 4, 5 or more). In some embodiments, the duplex heat-destabilizing modification is located at positions 2-9, or preferably positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the duplex heat-destabilizing modification is located at position 5, 6, 7, or 8 from the 5' end of the antisense strand.

In some further embodiments, the duplex heat-destabilizing modification is located at position 7 from the 5' end of the antisense strand.

The term "thermally destabilizing modification" refers to a modification that results in a decreased overall melting temperature (Tm) of dsRNA, preferably 1, 2, 3, or 4 degrees lower than that of dsRNA without such modification. In some embodiments, the duplex heat-destabilizing modification is at position 2, 3, 4, 5, 6, 7, 8 or 9 from the 5' end of the antisense strand. do.

。 Thermodestabilizing modifications include abasic modifications; mismatches with opposing nucleotides in opposing strands; and 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNA) or glycol nucleic acids (GNA), or 5 Contains sugar modifications such as '-2' linked nucleotides (e.g. those with 3'-OMe, 3'-F, 3'-H or 3'-OH, referred to herein as '3'-RNA') However, it is not limited to these. For example, thermodestabilizing modifications can include, but are not limited to, the following mUNA and GNA building blocks:

.

式中、Bはヌクレオチド塩基またはヌクレオチド塩基類似体であり、任意で、Bはアデニン、グアニン、シトシン、チミンまたはウラシルである。 In some embodiments, the destabilizing modification is one with a 5'-2' linked nucleotide (e.g., 3'-OMe, 3'-F, 3'-H or 3'-OH. , the destabilizing modification is a 5'-2' linked nucleotide with 3'-OMe, 3'-F, 3'-H or 3'-OH. In some embodiments, the destabilizing modification is A 5'-2' linked nucleotide with a 3'-OMe. In some embodiments, the destabilizing modification is a 5'-2' linked nucleotide with a 3'-F. In some embodiments, the destabilizing modification is a 5'-2' linked nucleotide with a 3'-F. The destabilizing modification is a 5'-2' linked nucleotide with a 3'-H. In some embodiments, the destabilizing modification is a 5'-2' linked nucleotide with a 3'-OH, e.g. The 2'-linked nucleotide is:

where B is a nucleotide base or nucleotide base analog, optionally B is adenine, guanine, cytosine, thymine or uracil.

In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA.

R＝H、OH;OMe;Cl、F;OH;O-（CH₂）₂OMe;SMe、NMe₂;NH₂;Me;CCH（アルキン）、O-nPr;O-アルキル;O-アルキルアミノ;
R'＝H、Me;
B＝A;C;5-Me-C;G;I;U;T;Y;2-チオウリジン;4-チオウリジン;C5-修飾ピリミジン;C2-修飾プリン;N8-修飾プリン;フェノキサジン;G-クランプ（G-clamp）;非標準単環式、二環式および三環式複素環;シュードウラシル;isoC;isoG;2,6-ジアミノプリン;シュードシトシン;2-アミノプリン;キサントシン;N6-アルキル-A;O6-アルキル-G;2-チオウリジン;4-チオウリジン;C5-修飾ピリミジン;C2-修飾プリン;N8-修飾プリン;7-デアザプリン、フェノキサジン;G-クランプ;非標準単環式、二環式および三環式複素環
からなる群より選択され、立体化学は、指定されていないキラル中心については、RまたはSならびにRおよびSの組合せである。 In some embodiments, the destabilizing modified mUNA is

R=H, OH;OMe;Cl, F;OH;O-(CH ₂ ) ₂ OMe;SMe, NMe ₂ ;NH ₂ ;Me;CCH (alkyne), O-nPr;O-alkyl;O-alkylamino ;
R'=H,Me;
B=A;C;5-Me-C;G;I;U;T;Y;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;phenoxazine;G- Clamp (G-clamp); non-standard monocyclic, bicyclic and tricyclic heterocycle; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl -A;O6-alkyl-G;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;7-deazapurine, phenoxazine;G-clamp;nonstandard monocyclic, di cyclic and tricyclic heterocycles, and the stereochemistry is R or S and combinations of R and S for unspecified chiral centers.

R＝H、OH;OMe;Cl、F;OH;O-（CH₂）₂OMe;SMe、NMe₂;NH₂;Me;CCH（アルキン）、O-nPr;O-アルキル;O-アルキルアミノ;
R'＝H、Me;
B＝A;C;5-Me-C;G;I;U;T;Y;2-チオウリジン;4-チオウリジン;C5-修飾ピリミジン;C2-修飾プリン;N8-修飾プリン;フェノキサジン;G-クランプ;非標準単環式、二環式および三環式複素環;シュードウラシル;isoC;isoG;2,6-ジアミノプリン;シュードシトシン;2-アミノプリン;キサントシン;N6-アルキル-A;O6-アルキル-G;2-チオウリジン;4-チオウリジン;C5-修飾ピリミジン;C2-修飾プリン;N8-修飾プリン;7-デアザプリン、フェノキサジン;G-クランプ;非標準単環式、二環式および三環式複素環
からなる群より選択され、立体化学は、指定されていないキラル中心については、RまたはSならびにRおよびSの組合せである。 In some embodiments, the destabilizing modified mUNA is

R=H, OH;OMe;Cl, F;OH;O-(CH ₂ ) ₂ OMe;SMe, NMe ₂ ;NH ₂ ;Me;CCH (alkyne), O-nPr;O-alkyl;O-alkylamino ;
R'=H,Me;
B=A;C;5-Me-C;G;I;U;T;Y;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;phenoxazine;G- Clamp; non-standard monocyclic, bicyclic and tricyclic heterocycle; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6- Alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard monocyclic, bicyclic and tricyclic is selected from the group consisting of the formula heterocycle and the stereochemistry is R or S and combinations of R and S for unspecified chiral centers.

R＝H、OMe;F;OH;O-（CH₂）₂OMe;SMe、NMe₂;NH₂;Me;O-nPr;O-アルキル;O-アルキルアミノ;
R'＝H、Me;
B＝A;C;5-Me-C;G;I;U;T;Y;2-チオウリジン;4-チオウリジン;C5-修飾ピリミジン;C2-修飾プリン;N8-修飾プリン;フェノキサジン;G-クランプ;非標準単環式、二環式および三環式複素環;シュードウラシル;isoC;isoG;2,6-ジアミノプリン;シュードシトシン;2-アミノプリン;キサントシン;N6-アルキル-A;O6-アルキル-G;7-デアザプリン
からなる群より選択され、立体化学は、指定されていないキラル中心については、RまたはSならびにRおよびSの組合せである。 In some embodiments, the destabilizing modified mUNA is

R=H, OMe;F;OH;O-(CH ₂ ) ₂ OMe;SMe, NMe ₂ ;NH ₂ ;Me;O-nPr;O-alkyl;O-alkylamino;
R'=H,Me;
B=A;C;5-Me-C;G;I;U;T;Y;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;phenoxazine;G- Clamp; non-standard monocyclic, bicyclic and tricyclic heterocycle; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6- alkyl-G;7-deazapurine and the stereochemistry is R or S and combinations of R and S for unspecified chiral centers.

R＝H、OH;OMe;Cl、F;OH;O-（CH₂）₂OMe;SMe、NMe₂;NH₂;Me;CCH（アルキン）、O-nPr;O-アルキル;O-アルキルアミノ;
R'＝H、Me;
B＝A;C;5-Me-C;G;I;U;T;Y;2-チオウリジン;4-チオウリジン;C5-修飾ピリミジン;C2-修飾プリン;N8-修飾プリン;フェノキサジン;G-クランプ;非標準単環式、二環式および三環式複素環;シュードウラシル;isoC;isoG;2,6-ジアミノプリン;シュードシトシン;2-アミノプリン;キサントシン;N6-アルキル-A;O6-アルキル-G;2-チオウリジン;4-チオウリジン;C5-修飾ピリミジン;C2-修飾プリン;N8-修飾プリン;7-デアザプリン、フェノキサジン;G-クランプ;非標準単環式、二環式および三環式複素環
からなる群より選択され、
立体化学は、指定されていないキラル中心については、RまたはSならびにRおよびSの組合せである。 In some embodiments, the destabilizing modified mUNA is

R=H, OH;OMe;Cl, F;OH;O-(CH ₂ ) ₂ OMe;SMe, NMe ₂ ;NH ₂ ;Me;CCH (alkyne), O-nPr;O-alkyl;O-alkylamino ;
R'=H,Me;
B=A;C;5-Me-C;G;I;U;T;Y;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;phenoxazine;G- Clamp; non-standard monocyclic, bicyclic and tricyclic heterocycle; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6- Alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard monocyclic, bicyclic and tricyclic selected from the group consisting of the formula heterocycle,
Stereochemistry is R or S and combinations of R and S for unspecified chiral centers.

R＝H、OH;OMe;Cl、F;OH;O-（CH₂）₂OMe;SMe、NMe₂;NH₂;Me;CCH（アルキン）、O-nPr;O-アルキル;O-アルキルアミノ;
R'＝H、Me;
B＝A;C;5-Me-C;G;I;U;T;Y;2-チオウリジン;4-チオウリジン;C5-修飾ピリミジン;C2-修飾プリン;N8-修飾プリン;フェノキサジン;G-クランプ;非標準単環式、二環式および三環式複素環;シュードウラシル;isoC;isoG;2,6-ジアミノプリン;シュードシトシン;2-アミノプリン;キサントシン;N6-アルキル-A;O6-アルキル-G;2-チオウリジン;4-チオウリジン;C5-修飾ピリミジン;C2-修飾プリン;N8-修飾プリン;7-デアザプリン、フェノキサジン;G-クランプ;非標準単環式、二環式および三環式複素環
からなる群より選択され、立体化学は、指定されていないキラル中心については、RまたはSならびにRおよびSの組合せである。 In some embodiments, the destabilizing modified mUNA is

R=H, OH;OMe;Cl, F;OH;O-(CH ₂ ) ₂ OMe;SMe, NMe ₂ ;NH ₂ ;Me;CCH (alkyne), O-nPr;O-alkyl;O-alkylamino ;
R'=H,Me;
B=A;C;5-Me-C;G;I;U;T;Y;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;phenoxazine;G- Clamp; non-standard monocyclic, bicyclic and tricyclic heterocycle; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6- Alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard monocyclic, bicyclic and tricyclic is selected from the group consisting of the formula heterocycle and the stereochemistry is R or S and combinations of R and S for unspecified chiral centers.

R＝H、OMe;F;OH;O-（CH₂）₂OMe;SMe、NMe₂;NH₂;Me;O-nPr;O-アルキル;O-アルキルアミノ;
R'＝H、Me;
B＝A;C;5-Me-C;G;I;U;T;Y;2-チオウリジン;4-チオウリジン;C5-修飾ピリミジン;C2-修飾プリン;N8-修飾プリン;フェノキサジン;G-クランプ;非標準単環式、二環式および三環式複素環;シュードウラシル;isoC;isoG;2,6-ジアミノプリン;シュードシトシン;2-アミノプリン;キサントシン;N6-アルキル-A;O6-アルキル-G;7-デアザプリン
からなる群より選択され、立体化学は、指定されていないキラル中心については、RまたはSならびにRおよびSの組合せである。 In some embodiments, the modified mUNA is

R=H, OMe;F;OH;O-(CH ₂ ) ₂ OMe;SMe, NMe ₂ ;NH ₂ ;Me;O-nPr;O-alkyl;O-alkylamino;
R'=H,Me;
B=A;C;5-Me-C;G;I;U;T;Y;2-thiouridine;4-thiouridine;C5-modified pyrimidine;C2-modified purine;N8-modified purine;phenoxazine;G- Clamp; non-standard monocyclic, bicyclic and tricyclic heterocycle; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6- alkyl-G;7-deazapurine and the stereochemistry is R or S and combinations of R and S for unspecified chiral centers.

式中、R＝H、Me、EtまたはOMe、R'＝H、Me、EtまたはOMe、R”＝H、Me、EtまたはOMe

式中、Bは修飾または無修飾核酸塩基であり、各構造上のアスタリスクはR、Sまたはラセミのいずれかを表す。 Exemplary abasic modifications include, but are not limited to, the following modifications:

where R=H, Me, Et or OMe, R'=H, Me, Et or OMe, R"=H, Me, Et or OMe

where B is a modified or unmodified nucleobase, and the asterisk on each structure represents either R, S or racemic.

式中、Bは修飾または無修飾核酸塩基であり、各構造上のアスタリスクはR、Sまたはラセミのいずれかを表す。 Exemplary sugar modifications include, but are not limited to, the following modifications:

where B is a modified or unmodified nucleobase, and the asterisk on each structure represents either R, S or racemic.

In some embodiments, the duplex thermally destabilizing modification is selected from the mUNA and GNA building blocks described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA. In some further embodiments of this, the dsRNA molecules include GNA, 2'-OMe, 3'-OMe, 5'-Me, Hyp-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h'GNA (Mod A ~Mod K).

であり、式中、Bは修飾または無修飾核酸塩基であり、R1およびR2は、独立して、H、ハロゲン、OR3、またはアルキルであり、R3は、H、アルキル、シクロアルキル、アリール、アラルキル、ヘテロアリールまたは糖である）。「UNA」という用語は、糖の結合のいずれかが除去されてアンロックド「糖」残基を形成しているアンロックド非環式核酸を指す。一例において、UNAは、C1'-C4'間の結合（すなわちC1'炭素とC4'炭素の間の炭素-酸素-炭素共有結合）が除去されているモノマーも包含する。もう一つの例では、糖のC2'-C3'結合（すなわちC2'炭素とC3'炭素の間の炭素-炭素共有結合）が除去される（Mikhailov et.al.,Tetrahedron Letters,26（17）:2059（1985）およびFluiter et al.,Mol.Biosyst.,10:1039（2009）参照。これらの文献は参照によりそれらの全体が本明細書に組み入れられる）。非環式誘導体は、ワトソン-クリック対合に影響を及ぼすことなく、バックボーンのフレキシビリティを増大させる。非環式ヌクレオチドは2'-5'連結部または3'-5'連結部によって連結されうる。 The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, such as any linkage between the ribose carbons (e.g. C1'-C2', C2'-C3', C3'-C4', C4'-O4' or C1'-O4') is absent and/or at least one of the ribose carbons or ribose oxygens (e.g. C1', C2', C3', C4' or O4') is independent Refers to deletions from nucleotides as a single or in combination. In some embodiments, the acyclic nucleotide is

where B is a modified or unmodified nucleobase, R1 and R2 are independently H, halogen, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl , heteroaryl or sugar). The term "UNA" refers to an unlocked acyclic nucleic acid in which any of the sugar linkages have been removed to form an unlocked "sugar" residue. In one example, UNA also includes monomers in which the C1'-C4' bond (ie, the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e. the carbon-carbon covalent bond between the C2' and C3' carbons) of the sugar is removed (Mikhailov et.al., Tetrahedron Letters, 26(17) Biosyst.:2059 (1985) and Fluiter et al., Mol. Biosyst., 10:1039 (2009), which are incorporated herein by reference in their entirety). Acyclic derivatives increase the flexibility of the backbone without affecting Watson-Crick pairing. Acyclic nucleotides can be linked by a 2'-5' linkage or a 3'-5' linkage.

を指す。 The term "GNA" refers to glycol nucleic acids, which are polymers similar to DNA or RNA, but whose "backbone" composition is composed of repeating glycerol units linked by phosphodiester bonds:

refers to

A thermodestabilizing modification of a duplex can be a mismatch (ie, a non-complementary base pair) between a thermodestabilizing nucleotide in the dsRNA duplex and the opposing nucleotide in the opposite strand. Exemplary mismatched base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T: T, U:T, or a combination thereof. Other mismatch base pairings known in the art are also applicable to the present invention. Mismatches can occur between nucleotides that are either natural or modified nucleotides. That is, mismatch base pairing can occur between the nucleobases of each nucleotide, regardless of the modification on the ribose sugar of the nucleotide. In certain embodiments, the dsRNA molecule contains at least one nucleobase in mismatched pairing that is a 2'-deoxynucleobase, eg, the 2'-deoxynucleobase is in the sense strand.

を含む。 In some embodiments, the duplex heat-destabilizing modification in the seed region of the antisense strand includes nucleotides that are impaired in WCH binding to complementary bases on the target mRNA, e.g.

including.

Further examples of abasic nucleotide modifications, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are detailed in WO 2011/133876, which is incorporated herein by reference in its entirety. .

Thermodestabilizing modifications can also include universal bases that have reduced or eliminated ability to hydrogen bond with opposing bases, and phosphate modifications.

である。 In some embodiments, thermally destabilizing modifications of the duplex include nucleotides with non-canonical bases, such as, but not limited to, the ability to form hydrogen bonds with bases in the opposite strand is impaired or completely This includes nucleobase modifications that are missing. These nucleobase modifications have been evaluated for destabilization of the central region of dsRNA duplexes as described in WO 2010/0011895, which is incorporated herein by reference in its entirety. . Exemplary nucleobase modifications include:

It is.

を含み、式中、Rは、H、OH、OCH₃、F、NH₂、NHMe、NMe₂またはO-アルキルである。 In some embodiments, the duplex heat-destabilizing modification in the seed region of the antisense strand includes one or more α-nucleotides complementary to bases on the target mRNA, e.g.

wherein R is H, OH, _OCH3 , F, _NH2 , NHMe, _NMe2 or O-alkyl.

である。 Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages include:

It is.

Alkyl as R group can be C ₁ -C ₆ alkyl. Specific alkyls as R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

Note that heat-destabilizing modifications can replace 2'-deoxynucleotides in the antisense strand. For example, the 2'-deoxynucleotides at positions 2, 5, 7, 12, 14 and/or 16 counting from the 5' end of the antisense strand can be replaced with thermodestabilizing modifications as described herein. Thus, in some embodiments, the antisense strand comprises 1, 2, 3, 4, 5 and 1 of positions 2, 5, 7, 12, 14 and/or 16 counting from the 5' end of the antisense strand. and/or six contain thermally destabilizing modifications. For example, the antisense strand contains thermodestabilizing modifications at positions 5 and 7 counting from the 5' end of the antisense strand.

In addition to the antisense strand containing a thermodestabilizing modification, the dsRNA can also contain one or more stabilizing modifications. For example, the dsRNA can include at least two (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications. Without limitation, stabilizing modifications can all be present in one strand. In some embodiments, both the sense and antisense strands contain at least two stabilizing modifications. Stabilizing modifications can be present on any nucleotide of the sense or antisense strand. For example, a stabilizing modification can be present on every nucleotide on the sense strand and/or on the antisense strand, or each stabilizing modification can be present on the sense strand or on the antisense strand in an alternating pattern; Alternatively, the sense or antisense strand contains both stabilizing modifications in an alternating pattern. The alternating pattern of stabilizing modifications on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of stabilizing modifications on the sense strand is the same as the alternating pattern of stabilizing modifications on the antisense strand. It may also be shifted.

In some embodiments, the antisense strand includes at least two (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications. Without limitation, stabilizing modifications in the antisense strand can be present at any position. In some embodiments, the antisense includes stabilizing modifications at positions 2, 6, 8, 9, 14 and 16 from the 5' end. In some other embodiments, the antisense includes stabilizing modifications at positions 2, 6, 14 and 16 from the 5' end. In some further embodiments, the antisense includes stabilizing modifications at positions 2, 14 and 16 from the 5' end.

In some embodiments, the antisense strand includes at least one stabilizing modification adjacent to the destabilizing modification. For example, a stabilizing modification can be a nucleotide at the 5' or 3' end of the destabilizing modification, ie, a nucleotide at the -1 or +1 position from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at the 5' and 3' ends of the destabilizing modification, i.e., at positions -1 and +1 from the position of the destabilizing modification, respectively. .

In some embodiments, the antisense strand has at least two stabilizing modifications at the 3' end of the destabilizing modification, i.e. at least two stabilizing modifications at positions +1 and +2 from the position of the destabilizing modification. including. In some embodiments, the sense strand includes at least two (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications. Without limitation, stabilizing modifications in the sense strand can be present at any position. In some embodiments, the sense strand includes stabilizing modifications at positions 7, 10 and 11 from the 5' end. In some other embodiments, the sense strand includes stabilizing modifications at positions 7, 9, 10 and 11 from the 5' end. In some embodiments, the sense strand includes stabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand includes stabilizing modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand includes a block of two, three or four stabilizing modifications.

In some embodiments, the sense strand does not include a stabilizing modification at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

Exemplary thermostabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermostabilizing modifications include, but are not limited to, LNA.

Note that thermostabilizing modifications can replace 2'-fluoronucleotides in the sense and/or antisense strands. For example, the 2'-fluoronucleotides at positions 8, 9, 10, 11 and/or 12 counting from the 5' end of the sense strand can be replaced with thermostabilizing modifications. Similarly, the 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand can be replaced with a thermostabilizing modification.

For a dsRNA molecule to be more effective in vivo, the antisense strand must have some degree of metabolic stability. In other words, for a dsRNA molecule to be more effective in vivo, some amount of the antisense strand may need to be present in vivo after a period of time after administration. Thus, in some embodiments, at 5 days after in vivo administration, at least 40%, such as at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% of the antisense strand of the dsRNA; At least 75% or at least 80% is present in vivo, for example in mouse liver. In some embodiments, at 6 days after in vivo administration, at least 40%, such as at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% is present in vivo, eg in mouse liver. In some embodiments, at 7 days after in vivo administration, at least 40%, such as at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% is present in vivo, eg in mouse liver. In some embodiments, at day 8 after in vivo administration, at least 40%, such as at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% is present in vivo, eg in mouse liver. In some embodiments, at 9 days after in vivo administration, at least 40%, such as at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% is present in vivo, eg in mouse liver. In some embodiments, at 10 days after in vivo administration, at least 40%, such as at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% is present in vivo, eg in mouse liver. In some embodiments, at day 11 after in vivo administration, at least 40%, such as at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% is present in vivo, eg in mouse liver. In some embodiments, at 12 days after in vivo administration, at least 40%, such as at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% is present in vivo, eg in mouse liver. In some embodiments, at 13 days after in vivo administration, at least 40%, such as at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% is present in vivo, eg in mouse liver. In some embodiments, at 14 days after in vivo administration, at least 40%, such as at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% is present in vivo, eg in mouse liver. In some embodiments, at 15 days after in vivo administration, at least 40%, such as at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% is present in vivo, eg in mouse liver.

Use of dsRNA The present invention further relates to the use of dsRNA molecules as defined herein for inhibiting the expression of target genes. In some embodiments, the invention further relates to the use of dsRNA molecules to inhibit target gene expression in vitro.

The invention further relates to dsRNA molecules as defined herein for use in inhibiting expression of a target gene in a subject. The subject can be any animal, such as a mammal, such as a mouse, rat, sheep, cow, dog, cat, or human.

In some embodiments, the dsRNA molecules of the invention are administered in a buffer.

In some embodiments, the siRNA compounds described herein can be formulated for administration to a subject. A formulated siRNA composition can be in a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (eg, less than 80, 50, 30, 20, or 10% water). In another example, the siRNA is in an aqueous phase, eg, in a solution containing water.

The aqueous phase or crystalline composition can be incorporated, for example, into a delivery vehicle, such as a liposome (particularly for an aqueous phase) or a particle (eg, a microparticle such as may be suitable for a crystalline composition). Generally, siRNA compositions are formulated in a manner compatible with the intended method of administration, as described herein. For example, in certain embodiments, the composition is prepared by at least one of the following methods: spray drying, freeze drying, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or Sonication, freeze-drying, condensation and other self-assembly with lipids.

The dsRNA preparation can be formulated in combination with another agent, such as another therapeutic agent, or with an agent that stabilizes the dsRNA, such as a protein that complexes with the dsRNA to form an iRNP. Still other agents include chelating agents, such as EDTA (eg, to remove divalent cations such as Mg ²⁺ ), salts, RNAse inhibitors (eg, broad-spectrum RNAse inhibitors such as RNAsin), and the like.

In some embodiments, the dsRNA preparation includes another dsRNA compound, such as a second dsRNA that can mediate RNAi with respect to a second gene or with respect to the same gene. Still other preparations can contain at least 3, 5, 10, 20, 50 or 100, or more different siRNA species. Such dsRNAs can mediate RNAi for a similar number of different genes.

In some embodiments, the dsRNA preparation comprises at least a second therapeutic agent (eg, an agent other than RNA or DNA). For example, dsRNA compositions for treating viral diseases, such as HIV, may include known antiviral agents, such as protease inhibitors or reverse transcriptase inhibitors. In another example, a dsRNA composition for treating cancer may further include a chemotherapeutic agent.

Exemplary formulations that can be used to administer the dsRNA molecules of the invention are discussed below.

Liposome. dsRNA preparations can be formulated into membrane molecular assemblies, such as liposomes or micelles, for delivery. The term "liposome" as used herein refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, such as a bilayer or multiple bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from lipophilic material and an aqueous interior. The aqueous portion contains the siRNA composition. The lipophilic material isolates the aqueous interior from the aqueous exterior, which typically does not contain an siRNA composition, but in some instances may contain an siRNA composition. Liposomes are useful for importing and delivering active ingredients to the site of action. Liposome membranes are structurally similar to biological membranes, so when liposomes are applied to tissues, the liposome bilayer fuses with the cell membrane bilayer. As liposome-cell coalescence progresses, the inner aqueous content containing dsRNA is delivered into the cell, where the dsRNA can specifically bind to target RNA and mediate RNAi. In some cases, liposomes are also specifically targeted, eg, to direct dsRNA to a particular cell type.

Liposomes containing dsRNA can be prepared by a variety of methods. In some examples, the lipid component of the liposome is dissolved in the detergent such that micelles are formed with the lipid component. For example, the lipid component can be an amphiphilic cationic lipid or a lipid conjugate. Detergents can have high critical micelle concentrations and can be non-ionic. Exemplary detergents include cholic acid, CHAPS, octyl glucoside, deoxycholic acid and lauroyl sarcosine. The dsRNA preparation is then added to the micelles containing the lipid component. Cationic groups on the lipids interact with the siRNA and condense around the dsRNA, forming liposomes. After condensation, the detergent is removed, such as by dialysis, to obtain a liposome preparation of dsRNA.

If necessary, carrier compounds that aid condensation can be added during the condensation reaction, such as by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid, such as spermine or spermidine. The pH can also be adjusted to favor condensation.

Further descriptions of methods for producing stable polynucleotide delivery vehicles incorporating polynucleotide/cationic lipid complexes as components of the delivery vehicle are described, for example, in WO96/37194. Liposome formation is described by Felgner, P.L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987, U.S. Patent No. 4,897,355, U.S. Patent No. 5,171,678, Bangham, et al. M. Mol. Biol.23:238,1965, Olson,et al.Biochim.Biophys.Acta 557:9,1979,Szoka,et al.Proc.Natl.Acad.Sci.75:4194,1978,Mayhew,et al.Biochim. Biophys.Acta 775:169,1984, Kim, et al.Biochim.Biophys.Acta 728:339,1983 and Fukunaga, et al.Endocrinol.115:757,1984. Aspects may also be included, which documents are incorporated herein by reference in their entirety. Techniques commonly used to prepare lipid aggregates of suitable size for use as delivery vehicles include sonication and freeze-thaw and extrusion (e.g. Mayer, et al. Biochim. Biophys. Acta 858: 161, 1986, which is incorporated herein by reference in its entirety). If consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidic techniques can be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984, this article (incorporated herein by reference in its entirety). These methods can be easily adapted to packaging siRNA preparations into liposomes.

pH-sensitive or negatively charged liposomes do not form complexes with nucleic acid molecules, but rather encapsulate them. Nucleic acid molecules and lipids are both similarly charged, so repulsion occurs rather than complex formation. Some nucleic acid molecules are nevertheless encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cultured cell monolayers. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274, incorporated herein by reference in its entirety).

One of the major types of liposome compositions contains phospholipids other than naturally occurring phosphatidylcholines. Neutral liposome compositions can be formed from, for example, dimyristoyl phosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and/or phosphatidylcholines and/or cholesterol.

Examples of other methods for introducing liposomes into cells in vitro include US Pat. No. 5,283,185, US Pat. 269:2550,1994, Nabel,Proc.Natl.Acad.Sci.90:11307,1993, Nabel,Human Gene Ther.3:649,1992, Gershon,Biochem.32:7143,1993 and Strauss EMBO J.11: 417,1992.

In some embodiments, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse to cell membranes. Although non-cationic liposomes cannot efficiently fuse with the plasma membrane, they are taken up by macrophages in vivo and can be used to deliver siRNA to macrophages.

Additional advantages of liposomes include that liposomes derived from natural phospholipids are biocompatible and biodegradable, liposomes can incorporate a wide range of water- and lipid-soluble drugs; Encapsulated siRNA can be protected from metabolism and degradation (Rosoff, "Pharmaceutical Dosage Forms", Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are lipid surface charge, vesicle size and liposome water volume.

Using the positively charged synthetic cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), it spontaneously interacts with nucleic acids to bind lipid- Small liposomes can be formed that form nucleic acid complexes, which by fusing with the negatively charged lipids of the plasma membrane of tissue culture cells, have the consequent ability to deliver siRNA (DOTMA and DNA For its use with, see, for example, Felgner, P.L. et al., Proc. (incorporated herein in its entirety).

The DOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA-complexed vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, MD) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells and is a negatively charged polypeptide. Contains positively charged DOTMA liposomes that spontaneously interact with nucleotides to form complexes. If enough positively charged liposomes are used, the net charge of the resulting complex will also be positive. The positively charged complexes prepared in this manner spontaneously attach to negatively charged cell surfaces and fuse with the plasma membrane to efficiently deliver functional nucleic acids, eg, into tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, IN), has an oleoyl moiety. It differs from DOTMA in that it is linked by an ester rather than an ether linkage.

Other cationic lipid compounds that have been reported include those conjugated to various moieties, such as carboxyspermine conjugated to one of two types of lipids, including 5-carboxyspermylglycine di Some include compounds such as octaoleylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (“DPPES”) (e.g., U.S. Pat. 5,171,678).

Another cationic lipid conjugate includes the derivatization of lipids with cholesterol (“DC-Chol”) formulated into liposomes in combination with DOPE (e.g., Gao, X. and Huang, L., Biochim.Biophys.Res.Commun.179:280, 1991). Lipopolylysine prepared by conjugating polylysine to DOPE has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991, which document is incorporated herein by reference in its entirety). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and result in more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for delivery of oligonucleotides are described in WO98/39359 and WO96/37194.

Liposomal formulations are particularly suitable for topical administration. Liposomes have several advantages compared to other formulations. Their advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer siRNA to the skin. In some embodiments, liposomes are used to deliver siRNA to epidermal cells and to enhance penetration of siRNA into dermal tissues, such as into the skin. For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes has been detailed (e.g. Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259 -265, Mannino, R.J. and Fould-Fogerite, S.,Biotechniques 6:682-690,1988, Itani,T.et al.Gene 56:267-276.1987, Nicolau,C.et al.Meth.Enz.149: 157-176, 1987, Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983, Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84: 7851-7855, 1987, which documents are incorporated herein by reference in their entirety).

Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been investigated to determine their utility in delivering drugs to the skin. Non-ionic liposome formulations containing Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) have been shown to be effective in the dermis of mouse skin. used to deliver drugs. Such formulations containing the dsRNA described herein are useful for treating skin disorders.

Liposomes containing dsRNA described herein can be made highly deformable. Such deformability may allow liposomes to penetrate through pores that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposome. Transfersomes can be created by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing dsRNA described herein can be delivered, eg, subcutaneously, by infection to deliver dsRNA to keratinocytes of the skin. To cross intact mammalian skin, lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of an appropriate transdermal gradient. In addition, due to the properties of lipids, these transfersomes can be self-optimizing (adaptive to the shape of the pore, e.g. in the skin), self-repairing, and in many cases , can reach their respective targets without fragmentation, and can often be self-loading.

Other formulations suitable for the present invention include U.S. Provisional Application No. 61/018,616, filed January 2, 2008; No. 61/039,748 filed on April 22, 2008, No. 61/047,087 filed on April 22, 2008, and No. 61/051,528 filed on May 8, 2008. . PCT Application No. PCT/US2007/080331 filed October 3, 2007 also describes formulations suitable for the present invention.

surfactant. The dsRNA composition can include a surfactant. In some embodiments, the dsRNA is formulated as an emulsion that includes a surfactant. The most common way to classify and rank the properties of the many different types of surfactants, both natural and synthetic, is through the use of hydrophilic/lipophilic balance (HLB). The nature of the hydrophilic group provides the most useful means of categorizing the various surfactants used in formulations (Rieger, "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988). p.285).

If a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have wide application in pharmaceutical products and can be used over a wide range of pH values. Generally, their HLB values range from 2 to about 18, depending on the respective structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters and ethoxylated esters. Also included in this class are nonionic alkanolamides and alkanol ethers, such as fatty alcohol ethoxylates, propoxylated alcohols and ethoxylated/propoxylated block polymers. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

A surfactant is classified as anionic if the surfactant molecule has a negative charge when dissolved or dispersed in water. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acylamides of amino acids, esters of sulfuric acids such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates and Includes sulfosuccinates, as well as phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

A surfactant is classified as cationic if the surfactant molecule has a positive charge when dissolved or dispersed in water. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most used members of this class.

If a surfactant molecule can have either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines and phosphatides.

The use of surfactants in drug products, formulations and emulsions has been reviewed (Rieger, "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

Micelles and other membrane formulations. For ease of explanation, micelles and other formulations, compositions and methods in this section generally discuss unmodified siRNA compounds. However, these micelles and other formulations, compositions and methods can also be practiced with other siRNA compounds, such as modified siRNA compounds, and such practice can be understood to be within the invention. siRNA compounds, such as double-stranded siRNA compounds, or ssiRNA compounds, such as larger siRNA compounds, or siRNA compounds, such as double-stranded siRNA compounds, or ssiRNA compounds, which can be processed into precursors, such as ssiRNA compounds, or precursors thereof; DNA encoding )), the composition can be provided as a micelle formulation. "Micelle" is defined herein as an amphiphilic molecule formed into a spherical structure such that all the hydrophobic parts of the molecule face inward and the hydrophilic part remains in contact with the surrounding aqueous phase. It is defined as a specific type of molecular assembly that is arranged. The opposite configuration exists if the environment is hydrophobic.

Mixed micelle formulations suitable for delivery by transdermal membranes can be prepared by mixing an aqueous solution of a dsRNA composition, an alkali metal C ₈ -C ₂₂ alkyl sulfate, and a micelle-forming compound. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, boridioil, evening primrose oil, menthol, trihydroxyoxocolanylglycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogs, Included are polidocanol alkyl ethers and their analogs, chenodeoxycholic acid, deoxycholic acid, and mixtures thereof. The micelle-forming compound can be added simultaneously with the addition of the alkali metal alkyl sulfate or after the addition of the alkali metal alkyl sulfate. Mixed micelles are formed by mixing virtually any type of component, but vigorous mixing is used to obtain micelles of smaller size.

In some methods, a first micelle composition is prepared that includes a dsRNA composition and at least an alkali metal alkyl sulfate. The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, a micelle composition is prepared by mixing the dsRNA composition, an alkali metal alkyl sulfate, and at least one micelle-forming compound, then adding the remaining micelle-forming compound and stirring vigorously.

Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added along with the micelle-forming components. Tonicity agents such as glycerin may also be added after formation of the mixed micellar composition.

To deliver the micellar formulation as a spray, the formulation can be placed in an aerosol dispenser, which is filled with a propellant. The propellant under pressure is liquid in the dispenser. The ratio of the components is adjusted such that there is one aqueous phase and one propellant phase, ie there is only one phase. If two phases are present, it may be necessary to shake the dispenser before dispensing a portion of the contents, eg through a metering valve. The dosage of the pharmaceutical agent is ejected as a fine mist from the metered valve.

Propellants can include hydrogenated chlorofluorocarbons, hydrogenated fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA134a (1,1,1,2 tetrafluoroethane) may be used.

Specific concentrations of essential components can be determined by relatively simple experimentation. For absorption through the oral cavity, it is often desirable to increase the dosage by, for example, at least two or three times when administered by injection or via the gastrointestinal route.

particle. In some embodiments, the dsRNA preparation can be incorporated into particles, such as microparticles. Microparticles can be produced by spray drying, but also by other methods such as freeze drying, evaporation, fluid bed drying, vacuum drying or a combination of these techniques.

Pharmaceutical Compositions The dsRNA agents of the invention can be formulated for pharmaceutical use. The present invention further relates to pharmaceutical compositions comprising dsRNA molecules as defined herein. Pharmaceutically acceptable compositions can be administered alone or formulated with one or more pharmaceutically acceptable carriers (excipients), excipients and/or diluents. , comprising a therapeutically effective amount of one or more dsRNA molecules of any of the above embodiments.

Pharmaceutical compositions can be specially formulated for administration as solids or liquids, including those compatible with the following administrations: (1) Oral administration, e.g. drinks (aqueous or non-aqueous solutions or suspensions), tablets, e.g. intended for oral mucosal, sublingual and systemic absorption, boluses, powders, granules, on the tongue. (2) parenteral administration, e.g. by subcutaneous, intramuscular, intravenous or epidural injection as a sterile solution or suspension or sustained release formulation; (3) external application; (4) intravaginal or rectal administration, e.g. as a pessary, cream or foam; (5) sublingual administration; 6) ocular administration, (7) transdermal administration, or (8) nasal administration. Delivery using subcutaneous or intravenous methods is particularly advantageous.

As used herein, the phrase "therapeutically effective amount" is the amount of a compound, material or composition comprising a compound of the invention that is effective in any medical treatment in at least a subpopulation of cells in an animal. It means an amount effective to produce any desired therapeutic effect at an applicable reasonable benefit/risk ratio.

The term "pharmaceutically acceptable" as used herein means that, within the scope of sound medical judgment, there is no risk of excessive toxicity, irritation, allergic response or other problems commensurate with a reasonable benefit/risk ratio. or used to refer to compounds, materials, compositions, and/or dosage forms that are suitable for use in contact with human and animal tissue without complications.

As used herein, the phrase "pharmaceutically acceptable carrier" refers to any pharmaceutically acceptable carrier necessary to transport or transport the compound from one organ or part of the body to another organ or part of the body. materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, manufacturing aids such as lubricants, magnesium talc, calcium or zinc stearate, or steric acid. )) or solvent-encapsulated material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose, (2) starches such as corn starch and potato starch, (3) cellulose and its like. Derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate, (4) tragacanth powder, (5) malt, (6) gelatin, (7) lubricants such as magnesium stearate, sodium lauryl sulfate and talc, ( 8) Excipients, such as cocoa butter and suppository waxes, (9) Oils, such as arachis oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, (10) Glycols, such as propylene glycol, ( 11) Polyols such as glycerin, sorbitol, mannitol and polyethylene glycol, (12) Esters such as ethyl oleate and ethyl laurate, (13) Agar, (14) Buffers such as magnesium hydroxide and aluminum hydroxide, (15) ) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solution, (21) polyester, polycarbonate and/or polyanhydride, ( 22) Bulking agents such as polypeptides and amino acids, (23) Serum components such as serum albumin, HDL and LDL, and (22) Other non-toxic compatible substances used in pharmaceutical formulations.

The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending on the host treated, the particular mode of administration. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, this amount will range from about 0.1 percent to about 99 percent of active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent, out of 100 percent. .

In certain embodiments, the formulations of the invention contain excipients selected from the group consisting of cyclodextrins, cellulose, liposomes, micelle-forming agents such as bile acids, and polymeric carriers such as polyesters and polyanhydrides; Compounds of. In certain embodiments, the aforementioned formulations render the compounds of the invention orally bioavailable.

The dsRNA agent preparation can be formulated in combination with another agent, such as another therapeutic agent, or with an agent that stabilizes the dsRNA, such as a protein that complexes with the dsRNA to form an iRNP. Still other agents include chelating agents, such as EDTA (eg, to remove divalent cations such as Mg ²⁺ ), salts, RNAse inhibitors (eg, broad-spectrum RNAse inhibitors such as RNAsin), and the like.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the invention with the carrier and, optionally, one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately bringing into association a compound of the invention with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

In some cases, it is desirable to slow the absorption of a drug from subcutaneous or intramuscular injection in order to prolong the drug's effects. This may be achieved by the use of suspensions of crystalline or amorphous materials that are poorly water soluble. The rate of absorption of the drug then depends on its rate of dissolution, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered dosage forms is accomplished by dissolving or suspending the drug in an oil vehicle.

The compounds of the invention, by analogy with other pharmaceuticals, may be formulated for administration in any convenient manner for use in medicine or veterinary medicine.

The term "treatment" is intended to include therapy and cure. Patients undergoing this treatment are any animal in need thereof, such as primates, especially humans, and other mammals, such as horses, cows, pigs and sheep, as well as poultry and pets in general.

Double-stranded RNA agents are produced in cells in vivo, eg, from an exogenous DNA template delivered into the cells. For example, a DNA template can be inserted into a vector and used as a gene therapy vector. Gene therapy vectors can be administered, for example, by intravenous injection, local administration (U.S. Pat. No. 5,328,470, herein incorporated by reference in its entirety), or stereotactic injection (see, for example, Chen et al. (1994) Proc. Natl.Acad.Sci.USA 91:3054-3057, herein incorporated by reference in its entirety). Pharmaceutical preparations of gene therapy vectors can include the gene therapy vector in an acceptable diluent or can include a slow release matrix in which the gene delivery vehicle is embedded. The DNA template may, for example, contain two transcription units, one transcription unit producing a transcript comprising the top strand of the dsRNA molecule and one transcription unit producing a transcript comprising the bottom strand of the dsRNA molecule. I can do it. Once those templates are transcribed, dsRNA molecules are generated and processed into siRNA agent fragments that mediate gene silencing.

A dsRNA molecule as defined herein or a pharmaceutical composition comprising a dsRNA molecule as defined herein can be administered to a subject using a variety of delivery routes. Compositions comprising dsRNA described herein can reach a subject by a variety of routes. Exemplary routes include intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

The dsRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more dsRNAs and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are compatible with the pharmaceutical administration. etc. shall be included. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional medium or agent is contemplated for use in the present compositions, so long as it is not incompatible with the active compound. Supplementary active compounds can also be incorporated into the compositions.

The compositions of the invention may be administered in a number of ways, depending on whether local or systemic treatment is desired, and depending on the area to be treated. Administration can be external (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intravenous administration.

The route and site of administration can be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscle of interest would be a logical choice. By administering dsRNA in aerosol form, lung cells can be targeted. Vascular endothelial cells can be targeted by coating a balloon catheter with dsRNA and mechanically introducing the dsRNA.

In one aspect, the invention features a method of administering a dsRNA molecule, eg, a dsRNA agent described herein, to a subject (eg, a human subject). In another aspect, the invention relates to a dsRNA molecule as defined herein for use in inhibiting expression of a target gene in a subject. The method or medical use involves administering a unit dose of a dsRNA molecule, such as a dsRNA agent described herein. In some embodiments, the unit dose is less than 10 mg per kg body weight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg body weight, and less than 200 nmoles of RNA agent (e.g. approximately 4.4 x ¹⁰ copies) per kg body weight, or 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075 RNA agent per kg body weight, Less than 0.0015, 0.00075, 0.00015 nmole.

The defined amount can be an amount effective to treat or prevent a disease or disorder, eg, a disease or disorder associated with the target gene. Unit doses can be administered, for example, by injection (eg, intravenously, subcutaneously or intramuscularly), by inhalation, or by topical application. In some embodiments, the dosage can be less than 10, 5, 2, 1 or 0.1 mg/kg body weight.

In some embodiments, the unit dose is administered less than once a day, such as less than once every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered at a certain frequency (eg, at a regular frequency). For example, a unit dose may be administered only once.

In some embodiments, the effective dose is administered in conjunction with other conventional treatment modalities. In some embodiments, the subject has a viral infection and the modality is an antiviral agent other than a dsRNA molecule, eg, other than an siRNA agent. In another embodiment, the subject has atherosclerosis and an effective amount of a dsRNA molecule, e.g., an siRNA agent, is subsequently administered, e.g. in combination with surgical intervention, e.g. in combination with angioplasty. .

In some embodiments, the subject has a dsRNA molecule encoding, e.g., a siRNA agent (e.g., a precursor, e.g., a larger dsRNA molecule that can be processed into an siRNA agent, or a dsRNA molecule, e.g., an siRNA agent, or a precursor thereof). An initial dose and one or more maintenance doses of DNA) are administered. The maintenance dose or doses may be the same as the initial dose or may be less than the initial dose, eg, half the initial dose. Maintenance regimens include treating subjects once or multiple times at doses ranging from 0.01 μg to 15 mg/kg body weight per day, such as 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg/kg body weight per day. may include doing. Maintenance doses are administered, for example, no more frequently than once every 2, 5, 10 or 30 days. Furthermore, the treatment regimen is continued over a period of time, which period will vary depending on the nature of the particular disease, its severity and the overall condition of the patient. In certain embodiments, dosages may be delivered no more frequently than once a day, such as no more than once every 24, 36 or 48 hours or more, such as no more than once every 5 or 8 days. Following treatment, the patient can be monitored for changes in their condition and for improvement in symptoms of the disease condition. The dosage of the compound may be increased if the patient does not respond significantly to the current dosage level, or an improvement in symptoms of the disease state is observed, the disease state disappears, or is undesirable. The dose can be reduced if side effects are observed.

The effective dose can be administered in a single dose or in two or more doses, as desired or as appropriate under the particular circumstances. If repeated or frequent infusions are desired, implantation of a delivery device, such as a pump, a semi-permanent stent (eg, intravenous, intraperitoneal, intracisternal or intrasaccular), or a reservoir, may be advantageous.

In some embodiments, the composition includes multiple dsRNA species. In another embodiment, the dsRNA species has a sequence that is neither overlapping nor contiguous with another species with respect to the natural target sequence. In another embodiment, the multiple dsRNA species are specific for different natural target genes. In another embodiment, the dsRNA molecule is allele specific.

The dsRNA molecules of the invention described herein can be administered to mammals, particularly large mammals, such as non-human primates or humans, in several ways.

In some embodiments, administration of the dsRNA molecule, e.g., siRNA agent, composition is via parenteral administration, e.g., intravenously (e.g., as a bolus or as a diffusible injection), intradermally, intraperitoneally, intramuscularly, intrathecally. , intracranial, intracranial, subcutaneous, transmucosal, oral mucosal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, or ocular administration. Administration can be performed by the subject or by another person, such as a health care provider. Dosing can be done in metered doses or with a dispenser that delivers metered doses. The chosen delivery mode is detailed below.

The present invention provides methods, compositions, and kits for rectal administration or delivery of dsRNA molecules described herein.

In a particular embodiment, the invention relates to a dsRNA molecule of the invention for use in the above method.

Methods of Inhibiting Target Gene Expression Aspects of the invention also relate to methods for inhibiting target gene expression. The method includes administering a dsRNA molecule of any of the above embodiments in an amount sufficient to inhibit expression of the target gene. The invention further relates to the use of a dsRNA molecule as defined herein for inhibiting the expression of a target gene in a target cell. In a preferred embodiment, the invention further relates to the use of dsRNA molecules to inhibit the expression of target genes in target cells in vitro.

In another aspect, the invention relates to a method of modulating the expression of a target gene in a cell, the method comprising providing the cell with a dsRNA molecule of the invention. In some embodiments, the target gene is Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK Gene, JNK gene, RAF gene, Erk1/2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepcidin, activated protein C, cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene, p73 Mutations in the gene, mutations in the p21 (WAF1/CIP1) gene, mutations in the p27 (KIP1) gene, mutations in the PPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIB I gene , a mutation in the MTAI gene, a mutation in the M68 gene, a mutation in the tumor suppressor gene, and a mutation in the p53 tumor suppressor gene.

In a particular embodiment, the invention relates to a dsRNA molecule of the invention for use in the above method.

Exemplary embodiments of various aspects may be described by the following embodiments with letter assignments.

Embodiment A: A dsRNA agent comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, the sense strand having 2 nucleotides at position 10 counting from the 5' end of the sense strand. '-fluoronucleotides, and the antisense strand contains 2'-deoxynucleotides at positions 5 and 7 counting from the 5' end of the antisense strand.

Embodiment B: The dsRNA agent according to embodiment A, wherein the sense strand further comprises a 2'-fluoronucleotide at position 11 counting from the 5' end of the sense strand.

Embodiment C: The dsRNA agent according to embodiment A or B, wherein the sense strand further comprises a 2'-fluoronucleotide at position 9 counting from the 5' end of the sense strand.

Embodiment D: dsRNA according to any one of embodiments AC, wherein the sense strand further comprises 2'-fluoronucleotides at positions 9 and 11 counting from the 5th end of the sense strand.

Embodiment E: The dsRNA agent according to any one of embodiments AD, wherein the sense strand comprises 2'-fluoronucleotides at positions 8 and 9 counting from the 5th end of the sense strand.

Embodiment F: The dsRNA agent according to any one of embodiments A to E, wherein the sense strand comprises 2'-fluoronucleotides at positions 11 and 12 counting from the 5th end of the sense strand.

Embodiment G: The dsRNA agent according to any one of embodiments A to F, wherein the sense strand comprises at least one 2'-OMe nucleotide.

Embodiment H: The dsRNA agent according to any one of embodiments AG, wherein the antisense strand comprises a 2'-deoxynucleotide at position 2 counting from the 5' end of the antisense strand.

Embodiment I: The dsRNA agent according to any one of embodiments A to H, wherein the antisense strand comprises 2'-deoxynucleotides at positions 2, 5 and 7 counting from the 5' end of the antisense strand.

Embodiment J: The dsRNA agent according to any one of embodiments AI, wherein the antisense strand comprises at least one 2'-fluoronucleotide.

Embodiment K: The dsRNA agent according to any one of embodiments A to J, wherein the antisense strand comprises a 2'-fluoronucleotide at position 14 of the antisense strand, counting from the 5' end of the antisense strand.

Embodiment L: The dsRNA agent according to any one of embodiments A to K, wherein the dsRNA agent comprises a ligand.

Embodiment M: The dsRNA agent according to any one of embodiments AL, wherein the sense strand comprises a ligand.

Embodiment N: The dsRNA agent according to embodiment L or M, wherein the ligand is an ASGPR ligand.

Embodiment O: The dsRNA agent according to any one of embodiments AN, comprising at least two phosphorothioate internucleotide linkages.

Embodiment P: The dsRNA agent according to any one of embodiments AO, wherein the sense strand comprises at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 5' end of the sense strand.

Embodiment Q: The antisense strand contains at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counted from the 5' end of the antisense strand, and between the first 5 nucleotides counted from the 3' end of the antisense strand. dsRNA agent according to any one of embodiments AP, comprising at least two phosphorothioate internucleotide linkages between the nucleotides.

Embodiment R: The dsRNA agent according to any one of embodiments A to Q, wherein the dsRNA has a double-stranded region of 18 to about 25 base pairs.

Embodiment S: The dsRNA agent according to any one of embodiments A to R, wherein the sense strand is 18 to 23 nucleotides in length.

Embodiment T: The dsRNA agent according to any one of embodiments A to S, wherein the antisense strand is 18 to 25 nucleotides in length.

Embodiment U: A dsRNA agent comprising a sense strand and an antisense strand, the sense strand being 18 to 23 nucleotides in length and comprising a 2'-fluoronucleotide at position 10 counting from the 5' end of the sense strand; The sense strand contains a 2'-fluoronucleotide at position 9 or 11, counting from the 5' end, and the antisense strand is 18 to 25 nucleotides long, and the antisense strand contains a 2'-fluoronucleotide at positions 5 and 7, counting from the 5' end of the antisense strand. The dsRNA agent comprises a 2'-deoxynucleotide.

Embodiment V: A dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand is 18 to 23 nucleotides in length and contains 2'-fluorocarbons at positions 9, 10 and 11 counting from the 5' end of the sense strand. nucleotides, and wherein the antisense is 18-25 nucleotides in length and includes 2'-deoxynucleotides at positions 5 and 7 counting from the 5' end of the antisense strand.

Further exemplary embodiments may be described by one or more of the numbered embodiments below.

Embodiment 1: A dsRNA agent comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, each nucleotide independently modified or unmodified,
the sense strand contains a 2'-fluoronucleotide at position 10 counting from the 5' end of the sense strand;
The antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7 and 12 counting from the 5' end of the antisense strand;
(i) the antisense strand contains a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand and a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, or (ii) the antisense strand contains a 2'-deoxynucleotide at position 14 or 16, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand; -contains nucleotides other than fluoronucleotides,
The dsRNA agent.

Embodiment 2: The dsRNA agent according to embodiment 1, wherein the sense strand further comprises a 2'-fluoronucleotide at position 11 counting from the 5' end of the sense strand.

Embodiment 3: The dsRNA agent according to embodiment 1 or 2, wherein the sense strand further comprises a 2'-fluoronucleotide at position 9 counting from the 5' end of the sense strand.

Embodiment 4: dsRNA according to any one of embodiments 1 to 3, wherein the sense strand further comprises 2'-fluoronucleotides at positions 9 and 11 counting from the 5th end of the sense strand.

Embodiment 5: The dsRNA agent according to any one of embodiments 1 to 4, wherein the sense strand comprises 2'-fluoronucleotides at positions 8 and 9 counting from the 5th end of the sense strand.

Embodiment 6: The dsRNA agent according to any one of embodiments 1 to 5, wherein the sense strand comprises 2'-fluoronucleotides at positions 11 and 12 counting from the 5th end of the sense strand.

Embodiment 7: The dsRNA agent according to any one of embodiments 1 to 6, wherein the sense strand comprises a nucleotide other than 2'-fluoro at position 7 counting from the 5' end of the sense strand.

Embodiment 8: The method of embodiments 1 to 7, wherein the sense strand comprises 2'-fluoronucleotides at positions 9, 10 and 11 counting from the 5' end of the sense strand, and a nucleotide other than 2'-fluoro at position 7. dsRNA according to any one of the items.

Embodiment 9: A dsRNA agent according to any one of embodiments 1 to 8, wherein the sense strand comprises at least one 2'-OMe nucleotide.

Embodiment 10: The dsRNA agent according to any one of embodiments 1 to 9, wherein the sense strand comprises a 2'-OMe nucleotide at position 7 counting from the 5' end of the sense strand.

Embodiment 11: Any of embodiments 1 to 10, wherein the sense strand comprises 2'-fluoronucleotides at positions 9, 10 and 11 counting from the 5' end of the sense strand, and a 2'-OMe nucleotide at position 7. dsRNA agent according to item 1.

Embodiment 12: The antisense strand comprises a 2'-fluoronucleotide at position 14 of the antisense strand counting from the 5' end of the antisense strand, and a nucleotide other than 2'-deoxy or 2'-fluoronucleotide at position 16. 12. The dsRNA agent according to any one of embodiments 1 to 11, comprising:

Embodiment 13: The antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7 and 12, a 2'-fluoronucleotide at position 14, and a 2'-fluoronucleotide at position 16, counting from the 5' end of the antisense strand. 13. The dsRNA agent according to any one of embodiments 1 to 12, wherein the dsRNA agent comprises a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide.

Embodiment 14: The dsRNA agent according to any one of embodiments 1 to 13, wherein the antisense strand comprises a 2'-OMe nucleotide at position 16 counting from the 5' end of the antisense strand.

Embodiment 15: The antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7 and 12 counting from the 5' end of the antisense strand, a 2'-fluoronucleotide at position 14, and a 2'-fluoronucleotide at position 16. 15. The dsRNA agent according to any one of embodiments 1 to 14, comprising a 2'-OMe nucleotide.

Embodiment 16: The antisense strand comprises a 2'-deoxynucleotide at position 14 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand comprises a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand. 12. The dsRNA agent according to any one of embodiments 1 to 11, comprising nucleotides other than 2'-fluoronucleotides.

Embodiment 17: The antisense strand comprises a 2'-deoxynucleotide at position 14 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand comprises a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand. 17. The dsRNA agent according to any one of embodiments 1-11 or 16, comprising a 2'-OMe nucleotide.

Embodiment 18: The antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12 and 14 of the antisense strand counting from the 5' end of the antisense strand, and the sense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from the 5' end of the 18. The dsRNA agent according to any one of embodiments 1 to 11 or 16 to 17, comprising a nucleotide other than a 2'-fluoronucleotide in position 7, counting from the end.

Embodiment 19: The antisense strand comprises a 2'-deoxynucleotide at position 16 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand comprises a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand. 19. The dsRNA agent according to any one of embodiments 1 to 18, comprising nucleotides other than 2'-fluoronucleotides.

Embodiment 20: The antisense strand comprises a 2'-deoxynucleotide at position 16 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand comprises a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand. 20. The dsRNA agent according to any one of embodiments 1 to 19, comprising a 2'-OMe nucleotide.

Embodiment 21: The antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12 and 16 of the antisense strand counting from the 5' end of the antisense strand, and the sense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12 and 16 of the antisense strand, counting from the 5' end of the 21. The dsRNA agent according to any one of embodiments 1 to 20, comprising a nucleotide other than 2'-fluoro in position 7, counting from the end.

Embodiment 22: The antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12, 14 and 16 of the antisense strand counting from the 5' end of the antisense strand, and the sense strand comprises dsRNA agent according to any one of embodiments 1 to 11 or 16 to 21, comprising a nucleotide other than 2'-fluoro in position 7 counting from the 5' end of the dsRNA agent.

Embodiment 23: The antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, 14 and 16, and the sense strand contains a 2'-fluoronucleotide at position 7 counting from the 5' end of the sense strand. dsRNA agent according to any one of embodiments 1 to 11 or 16 to 22, comprising a nucleotide other than nucleotides.

Embodiment 24: The antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12 and 16 of the antisense strand counting from the 5' end of the antisense strand and a 2'-fluoro at position 14. , and the sense strand comprises a nucleotide other than 2'-fluoro at position 7 counting from the 5' end of the sense strand.

Embodiment 25: The antisense strand comprises 2'-deoxynucleotides at positions 2, 5, 7, 12 and 16 of the antisense strand counting from the 5' end of the antisense strand and a 2'-fluoro at position 14. , and the sense strand comprises a 2'-OMe nucleotide at position 7 counting from the 5' end of the sense strand.

Embodiment 26: A dsRNA agent according to any one of embodiments 1 to 25, comprising a ligand.

Embodiment 27: A dsRNA agent according to any one of embodiments 1 to 26, wherein the sense strand comprises a ligand.

Embodiment 28: A dsRNA agent according to embodiment 27, wherein the ligand is at the 3' end of the sense strand.

Embodiment 29: A dsRNA agent according to embodiment 27, wherein the ligand is at the 5' end of the sense strand.

Embodiment 30: A dsRNA agent according to any one of embodiments 26 to 29, wherein the ligand comprises an ASGPR ligand.

Embodiment 31: A dsRNA agent according to any one of embodiments 26 to 29, wherein the ligand is a lipophilic group.

Embodiment 32: The dsRNA agent according to embodiment 31, wherein the ligand is a C _10-30 aliphatic group.

Embodiment 33: The dsRNA agent according to embodiment 32, wherein the C _10-30 aliphatic group is a C _10-30 alkyl group.

Embodiment 34: The dsRNA agent according to embodiment 33, wherein the C _10-30 alkyl group is a linear or branched tetradecyl, hexadecyl, octadecyl, icosyl, docosyl or tetracosyl group.

Embodiment 35: A dsRNA agent according to embodiment 27, wherein the ligand is conjugated to a non-terminal nucleotide of the sense strand.

Embodiment 36: The ligand is conjugated to the 2' position of the non-terminal nucleotide of the sense strand, optionally to one of positions 5, 6, 7 or 8 of the sense strand, counting from the 5' end. 36. The dsRNA agent according to embodiment 35.

Embodiment 37: The ligand comprises an abasic nucleotide, optionally the abasic nucleotide being an inverted nucleotide, linked to the strand of the dsRNA agent via a 5'→5' linkage or a 3'→3' linkage. 37. The dsRNA agent according to any one of embodiments 26 to 36, wherein the dsRNA agent is

Embodiment 38: A dsRNA agent according to any one of embodiments 26 to 37, wherein the ligand is attached to the 3' end of the sense strand.

Embodiment 39: A dsRNA agent according to embodiment 38, wherein the ligand is attached to the 3' end of the sense strand via a 3'→3' linkage.

Embodiment 40: A dsRNA agent according to any one of embodiments 1 to 39, wherein the dsRNA comprises two ligands.

Embodiment 41: The dsRNA according to embodiment 40, wherein the sense strand comprises a first ligand attached to the 3' end of the sense strand and a second ligand attached to the 5' end of the sense strand.

Embodiment 42: The first ligand comprises an abasic nucleotide, the second ligand comprises an ASGPR ligand, and optionally the abasic nucleotide is an inverted nucleotide, linked to the sense strand via a 3'→3' junction. dsRNA according to embodiment 41, wherein the dsRNA is

Embodiment 43: A dsRNA agent according to any one of embodiments 1 to 42, comprising at least two phosphorothioate internucleotide linkages.

Embodiment 44: A dsRNA agent according to any one of embodiments 1 to 43, wherein the sense strand comprises at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counting from the 5' end of the sense strand.

Embodiment 45: The antisense strand comprises at least two phosphorothioate internucleotide linkages between the first 5 nucleotides counted from the 5' end of the antisense strand, and between the first 5 nucleotides counted from the 3' end of the antisense strand. 45. The dsRNA agent according to any one of embodiments 1 to 44, comprising at least two phosphorothioate internucleotide linkages between the nucleotides.

Embodiment 46: The dsRNA agent according to any one of embodiments 1 to 45, wherein the dsRNA has a double-stranded region of 18 to about 25 base pairs.

Embodiment 47: A dsRNA agent according to any one of embodiments 1 to 46, wherein the sense strand is 18 to 23 nucleotides in length.

Embodiment 48: A dsRNA agent according to any one of embodiments 1 to 47, wherein the antisense strand is 18 to 25 nucleotides in length.

Embodiment 49: A dsRNA agent comprising a sense strand and an antisense strand, the sense strand being 18 to 23 nucleotides in length and comprising a 2'-fluoronucleotide at position 10 counting from the 5' end of the sense strand, The sense strand contains a 2'-fluoronucleotide at position 9 or 11, counting from the 5' end, and the antisense strand is 18 to 25 nucleotides long, and the antisense strand contains a 2'-fluoronucleotide at positions 2, 5, counting from the 5' end of the antisense strand. , 7 and 12 contain 2'-deoxynucleotides,
(i) the antisense strand contains a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand and a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, or (ii) the antisense strand contains a 2'-deoxynucleotide at position 14 or 16, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand; -contains nucleotides other than fluoronucleotides,
The dsRNA agent.

Embodiment 50: A dsRNA agent comprising a sense strand and an antisense strand, wherein the sense strand is 18 to 23 nucleotides long and contains 2'-fluorocarbons at positions 9, 10 and 11 counting from the 5' end of the sense strand. nucleotides, and the antisense is 18 to 25 nucleotides in length and includes 2'-deoxynucleotides at positions 2, 5, 7, and 12 counting from the 5' end of the antisense strand;
(i) the antisense strand contains a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand and a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, or (ii) the antisense strand contains a 2'-deoxynucleotide at position 14 or 16, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand; -contains nucleotides other than fluoronucleotides,
The dsRNA agent.

Embodiment 51: A dsRNA agent according to any one of embodiments 1 to 50, comprising a phosphate mimic at the 5' end of the antisense strand.

Embodiment 52: The dsRNA agent according to embodiment 51, wherein the phosphate mimic is 5'-E-vinylphosphonate.

を有する5'-シクロプロピルホスホネートであり、式中、＊は5'末端にあるヌクレオチドのC5位への結合である、態様52記載のdsRNA剤。 Aspect 53: The phosphate mimic has the structure:

53. The dsRNA agent according to embodiment 52, wherein the dsRNA agent is a 5'-cyclopropylphosphonate having the formula: where * is a bond to the C5 position of the nucleotide at the 5' end.

Embodiment 54: The remaining nucleotides in the sense strand (i.e. nucleotides in positions not otherwise defined) are unmodified nucleotides or modified nucleotides, optionally 2'-OMe, 2'-F, 2 Embodiments wherein the modified nucleotide is selected from the group consisting of '-H and 2'-OC _10-30 aliphatic groups, with the proviso that no more than one modified nucleotide is a 2'-OC _10-30 aliphatic group. dsRNA agent according to any one of 1 to 53.

Embodiment 55: The remaining nucleotides in the sense strand (i.e. the nucleotides in positions not otherwise defined) are 2'-OMe, 2'-F, 2'-H and 2'-OC _10-30 aliphatic groups. 55. The dsRNA agent according to any one of aspects 1 to 54, wherein the modified nucleotide is selected from the group consisting of, with the proviso that no more than one modified nucleotide is a 2'-OC _10-30 aliphatic group.

Embodiment 56: The remaining nucleotides in the antisense strand (i.e. nucleotides at positions not otherwise defined) are unmodified nucleotides or modified nucleotides, optionally 2'-OMe, 2'-F, a modified nucleotide selected from the group consisting of 2'-H, GNA and 3'-RNA, where 3'-RNA is optionally 3'-OH, provided that the modified nucleotide is GNA or 3'-RNA; 56. The dsRNA agent according to any one of embodiments 1 to 55, which is one or less.

Embodiment 57: The remaining nucleotides in the antisense strand (i.e. the nucleotides in positions not otherwise defined) are in the group consisting of 2'-OMe, 2'-F, 2'-H, GNA and 3'-RNA. wherein the 3'-RNA is optionally 3'-OH, with the proviso that no more than one modified nucleotide is GNA or 3'-RNA. dsRNA agents as described in Section.

Definitions Excerpt Certain terms used in the specification, examples, and appended claims are collected here for convenience. Unless otherwise stated or the context requires otherwise, the following terms and phrases shall have the meanings listed below. Unless explicitly stated otherwise or clear from the context, the following terms and phrases do not exclude the meanings that they have acquired in the relevant art. These definitions are included to help explain specific aspects and are not intended to limit the claimed invention. This is because the scope of the invention is limited only by the claims. Further, unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include the singular.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials can be used in the practice or testing of the present invention, such methods, devices, and materials are described herein in this regard.

Additionally, the practice of the present invention involves the use of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art, except as otherwise indicated. Conventional technology in science can be used. Such techniques are described in "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989), "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984), "Animal Cell Culture" (R.I. Freshney, ed. ., 1987), "Methods in Enzymology" (Academic Press, Inc.), "Current Protocols in Molecular Biology" (F.M. Ausubel et al., eds., 1987 and regularly updated editions), "PCR: The Polymerase Chain Reaction" , (Mullis et al., ed., 1994), “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988), and “Phage Display: A Laboratory Manual” (Barbas et al., 2001). detailed.

If a range of values is provided, each intervening value between the upper and lower limits of the range (up to one-tenth of the unit unless the context clearly indicates otherwise) and Any other stated or intervening values within that range are understood to be encompassed within the invention. The upper and lower limits of these subranges may be independently included in the subranges and are likewise encompassed by the invention, except for any specifically excluded limit within the stated range. Where the stated range includes one or both of the endpoints, ranges excluding either or both of those included limits are also included in the invention.

Certain ranges are presented herein in numerical terms preceded by the term "about." As used herein, the term "about" is used to literally support the exact number to which it is prefixed, as well as numbers that are near or approximate the number to which the term is prefixed. be done. In determining whether a number is near or approximates a specifically specified number, the near or approximate unspecified number is may be a number that is substantially equivalent to the number specified in .

As used herein, the term "comprising" or "comprises" is used in reference to compositions, methods, and their respective components that are essential, but not essential, to the invention. Inclusion of unspecified elements is also allowed regardless of the

The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" shall include "and" unless the context clearly indicates otherwise. Furthermore, it is noted that the claims may be drafted to exclude any element that is optional. Accordingly, this statement shall serve as a prerequisite basis for the use of exclusive expressions such as "only" or "only" or the use of "negative" limitations in connection with the specification of claim elements. .

As used herein, the terms "dsRNA", "siRNA" and "iRNA agent" refer to an agent capable of mediating the silencing of a target RNA, e.g. mRNA, e.g. the transcript of a gene encoding a protein. are used interchangeably to refer to. For convenience, such mRNA is also referred to herein as silenced mRNA. Such genes are also called target genes. Generally, the RNA to be silenced is an endogenous gene, an exogenous gene or a pathogenic gene. In addition, RNA other than mRNA can also be targeted, such as tRNA and viral RNA.

As used herein, the phrase "mediate RNAi" refers to the ability to sequence-specifically silence a target gene, eg, mRNA. Without wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and the antisense strand of a guide RNA, such as a dsRNA, where the antisense strand is 21 to 23 nucleotides long. It is long.

As used herein, "specifically hybridizable" and "complementary" refer to a sufficient degree of complementarity such that stable specific binding occurs between the present invention and the target RNA molecule. This is a term used for Specific binding refers to binding to a non-target sequence under conditions in which specific binding is desired, i.e., under physiological conditions in the case of an assay or therapeutic treatment, or under the conditions under which the assay is performed in the case of an in vitro assay. A sufficient degree of specificity is required to avoid non-specific binding of oligomeric compounds. Non-target sequences typically differ by at least 5 nucleotides.

In some embodiments, a dsRNA molecule of the invention is "sufficiently complementary" to a target RNA, such as a target mRNA, such that the dsRNA molecule silences production of a protein encoded by the target mRNA. In another embodiment, the dsRNA molecules of the invention are "strictly complementary" to the target RNA, e.g., by annealing of the target RNA and the dsRNA duplex agent, e.g. However, it forms hybrids made exclusively of Watson-Crick base pairs. A "sufficiently complementary" target RNA can include an internal region (eg, a region of at least 10 nucleotides) that is strictly complementary to the target RNA. Moreover, in some embodiments, the dsRNA molecules of the invention specifically discriminate between single nucleotide differences. In this case, the dsRNA molecule mediates RNAi only if strict complementarity is found in regions that differ by a single nucleotide (eg, within 7 nucleotides thereof).

。 The term "BNA" refers to cross-linked nucleic acids, often referred to as constrained or inaccessible RNA. BNAs can contain 5-, 6-, and even 7-membered bridge structures with "fixed" C ₃ '-endo sugar puckering. A bridge is typically incorporated at the 2',4' position of the ribose to provide a 2',4'-BNA nucleotide (eg LNA or ENA). Examples of BNA nucleotides include the following nucleosides:

.

に「ロック」することができる。 The term "LNA" refers to locked nucleic acid, often referred to as restricted RNA or inaccessible RNA. LNA is a modified RNA nucleotide. The ribose portion of the LNA nucleotide is modified with an additional bridge (eg, a methylene or ethylene bridge) that connects the 2' hydroxyl to the 4' carbon of the same ribose sugar. For example, the crosslink links the ribose in the 3'-endo (North) conformation:

can be “locked” to

The term "ENA" refers to ethylene-bridged nucleic acid (ethylene-bridged nucleic acid), often referred to as constrained or inaccessible RNA.

As used herein, "cleavage site" refers to a backbone junction in a target gene or sense strand that is cleaved by the RISC mechanism using an iRNA agent. The target cleavage site region also includes at least one or at least two nucleotides on either side of the cleavage site. In the case of the sense strand, if the sense strand itself is the target to be cleaved by the RNAi mechanism, the cleavage site is the backbone junction in the sense strand that is cleaved. Cleavage sites can be determined using methods known in the art, such as the 5'-RACE assay described in Soutschek et al., Nature (2004) 432, 173-178, which is incorporated herein by reference in its entirety. I can do it. As is well understood in the art, a conical double-stranded RNAi agent comprising two 21-nucleotide long strands (where the strands have a 2-nucleotide single-stranded overhang at the 3' end) (forming a double-stranded region of consecutive base pairs), the cleavage site region corresponds to positions 9-12 from the 5' end of the sense strand.

The terms "reduce," "reduce," "reduce," or "inhibit" are all used herein to refer to a decrease in a statistically significant amount. In some embodiments, "reducing", "reducing" or "decreasing" or "inhibiting" typically means at least 10 %, such as at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about May include a reduction of 99% or more. "Reduction" or "inhibition" as used herein does not encompass complete inhibition or complete reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to the reference level. The reduction is preferably to a level that is acceptable as within normal range for individuals without the given disorder.

As used herein, the "central region" of a chain refers to positions 5 to 17 counting from the 5' end of the chain, such as positions 6 to 16, positions 6 to 15, positions 6 to 14, positions 6 to 13, positions 6 ~12, position 7-15, position 7-14, position 7-13, position 7-12, position 8-16, position 8-15, position 8-14, position 8-13, position 8-12, position 9 -16, position 9-15, position 9-14, position 9-13, position 9-12, position 10-16, position 10-15, position 10-14, position 10-13 or position 10-12. For example, the central region of a chain refers to positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the chain. Preferred central regions of the sense strand are positions 6, 7, 8, 9, 10, 11, 12, 13 and 14 counting from the 5' end of the sense strand. More preferred central regions of the sense strand are positions 7, 8, 9, 10, 11, 12 and 13 counting from the 5' end of the sense strand. Preferred central regions of the antisense strand are positions 9, 10, 11, 12, 13, 14, 15, 16 and 17 counting from the 5' end of the antisense strand. More preferred central regions of the antisense strand are positions 10, 11, 12, 13, 14, 15 and 16 counting from the 5' end of the antisense strand.

It will be apparent to those skilled in the art upon reading this disclosure that each of the individual aspects described and illustrated herein has separate components and features, and that they do not fall within the scope or scope of the invention. It may readily be separated from or combined with any of the features of several other aspects without departing from the gist. Any of the methods presented may be performed in the order presented or in any other order that is logically possible.

The following examples are given to further illustrate the invention, but these examples should not be construed as further limitations. The contents of all references, pending patent applications and published patents cited throughout this application are hereby expressly incorporated by reference.

Oligonucleotide synthesis and purification All oligonucleotides were prepared on a 1 μmole scale on a MerMade 192 synthesizer using universal or custom supports. All phosphoramidites were used at a concentration of 100 mM in 100% acetonitrile or 9:1 acetonitrile:DMF with the standard protocol for 2-cyanoethyl phosphoramidites, but increasing the coupling time to 400 seconds. Oxidation of the newly formed linkage was performed using a solution of 50mM I2 in 9:1 acetonitrile:water to create a phosphate linkage or a solution of 100mM DDTT in 9:1 pyridine:acetonitrile to create a phosphorothioate linkage. Achieved using. After trityl-off synthesis, the column was incubated with 150 μL of 40% methylamine aqueous solution for 45 minutes and the solution was drained into a 96-well plate by vacuum. After repeated incubation and draining with fresh methylamine aqueous solution, the plate containing the crude oligonucleotide solution was sealed and shaken for an additional 60 min at room temperature to completely remove all protecting groups. Precipitation of crude oligonucleotides was achieved by adding 1.2 mL of 9:1 acetonitrile:EtOH to each well, followed by overnight incubation at -20°C. The plate was then centrifuged at 3000 RPM for 45 minutes, the supernatant was removed from each well, and the pellet was resuspended in 950 μL of 20 mM NaOAc aqueous solution. Finally, each crude solution was desalted on a GE Hi-Trap desalting column (Sephadex G25 Superfine) and water was used to elute the final oligonucleotide product. All identities and purity were confirmed by ESI-MS and IEX HPLC, respectively.

Cell Culture and Transfection Transfection of primary mouse or cynomolgus monkey hepatocytes (Thermo Fisher Scientific/Gibco) was performed in 384-well plates with 4.9 μL Opti-MEM + 0.1 μL Lipofectamine RNAiMax (Invitrogen, cat. no. 13778-150) per well. was performed by adding 5 μL of siRNA duplex to each well and incubating for 15 minutes at room temperature. Next, 40 μL of Dulbecco's modified Eagle's medium (PCH) or William's medium (PMH) containing approximately 5 x 103 cells was added to the siRNA mixture. Cells were incubated at 37°C for 24 hours before being processed for RNA purification. Experiments were performed with siRNA doses of 10 nM, 1 nM and 0.1 nM.

Parent dsRNA molecules are shown in Tables 1-3. Abbreviations used in the sequences are summarized in Table 4.

(Table 1) Sequence of parent dsRNA molecule

(Table 2) Additional exemplary sequences of parent dsRNA molecules

(Table 3) Additional exemplary sequences of parent dsRNA molecules

＊オリゴヌクレオチド中に存在する場合、これらのモノマーは5'-3'ホスホジエステル結合によって互いに連結されていることは理解されるだろう。また、ヌクレオチドが2'フルオロ修飾を含有する場合、そのフルオロは親ヌクレオチドのその位置にあるヒドロキシを置き換える（すなわち2'-デオキシ-2'-フルオロヌクレオチドである）と理解される。 (Table 4) Abbreviations of nucleotide monomers used in the description of nucleic acid sequences ^*

*It will be appreciated that when present in an oligonucleotide, these monomers are linked to each other by 5'-3' phosphodiester bonds. It is also understood that when a nucleotide contains a 2'fluoro modification, the fluoro replaces the hydroxy at that position of the parent nucleotide (ie, is a 2'-deoxy-2'-fluoronucleotide).

Some exemplary dsRNA molecule designs according to embodiments of the present disclosure are schematically shown in FIGS. 5A and 5B. Exemplary dsRNA molecules according to some embodiments of the present disclosure are listed in Tables 5 and 6.

Table 5: Exemplary dsRNA molecules according to some embodiments of the present disclosure

Table 6: Additional exemplary dsRNA molecules according to some embodiments of the present disclosure

In some embodiments, the dsRNA molecule is not a dsRNA molecule listed in Table 7.

(Table 7) Additional dsRNA molecules

Isolation of total RNA using the DYNABEADS mRNA Isolation Kit (Invitrogen™, part number 610-12) Cells were lysed in 75 μL of lysis/binding buffer containing 3 μL of beads per well and electrostatically Mixed for 10 minutes on an electrostatic shaker. The washing process was automated on a Biotek EL406 using a magnetic plate support. Beads were washed once in buffer A, once in buffer B, and twice in buffer E (in 90 μL) with an aspiration step in between. Following the final aspiration, a complete 10 μL RT mixture was added to each well as described below.

cDNA synthesis using the ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, catalog number 4368813)
A master mix of 1 uL 10x buffer, 0.4 μL 25x dNTPs, 1 μL random primer, 0.5 μL reverse transcriptase, 0.5 μL RNase inhibitor and 6.6 μL H ₂ O per reaction was added to each well. The plate was sealed and agitated on an electrostatic shaker for 10 minutes, then incubated at 37°C for 2 hours. Following that, the plate was stirred at 80°C for 8 minutes.

real-time PCR
In a master mix containing 0.5 μL human GAPDH TaqMan probe (4326317E), 0.5 μL human AGT (Hs00174854m1), 2 μL nuclease-free water and 5 μL Lightcycler 480 probe master mix (Roche catalog number 04887301001) per well of a 384-well plate, 2 μL of cDNA was added (Roche catalog number 04887301001). Real-time PCR was performed with a LightCycler480 real-time PCR system (Roche).

To calculate relative fold changes, data were analyzed using the ΔΔCt method and normalized to assays performed with 10 nM AD-1955-transfected or mock-transfected cells.

The results are shown in Figures 1A-1B and 3A-4C and summarized in Tables 8-14. These results demonstrate consistently improved in vitro activity compared to the parent design in several exemplary designs and in a large set of exemplary sequences, targets and cell lines.

(Table 8) In vitro activity of various designs in primary mouse hepatocytes

(Table 9) In vitro activity of various designs in primary mouse hepatocytes

(Table 10) In vitro activity of various designs in primary mouse hepatocytes

(Table 11) In vitro activity of various designs in primary mouse hepatocytes

(Table 12) In vitro activity of various designs in primary cynomolgus monkey hepatocytes

(Table 13) In vitro activity of various designs on Hep3B

(Table 14) In vitro activity of different designs targeting Agt in Hep3B

In Vivo Studies in Mice and Cynomolgus Monkeys All studies were conducted using protocols approved by Alnylam Pharmaceuticals' Institutional Animal Care and Use Committees (IACUCs), as appropriate, and consistent with local, state, and federal regulations.

For mouse pharmacodynamic studies, female C57BL/6 mice (Charles River Laboratories) were administered vehicle control (1× PBS or 0.9% sodium chloride) or a single dose of siRNA subcutaneously in the upper back. Blood was collected by retroorbital bleed. Serum was separated at 13000 rpm for 10 minutes at room temperature. Mouse livers were collected, immediately snap-frozen in liquid nitrogen, and stored at -80°C for mRNA and siRNA analysis.

As shown in Figures 2A-2D and Figures 10A-10D, these results demonstrate improved or similar target knockdown in mice and improved or similar target knockdown and duration of silencing in cynomolgus monkeys. It has been proven.

Serum protein quantification
TTR protein was quantified by ELISA from serum isolated from whole blood. ELISA was performed following the manufacturer's protocol (ALPCO, 41-PALMS-E01) after a 3025-fold dilution of serum samples. Data were normalized to pre-bleed TTR levels. All samples were assayed in duplicate. Each data point is the average of all mice within each cohort (n=3).

In some embodiments, the dsRNA molecule is not a dsRNA molecule listed in any one of Tables 15-25.

(Table 15) Exemplary dsRNA molecules

(Table 16) Exemplary dsRNA molecules

(Table 17) Exemplary dsRNA molecules

(Table 18) Exemplary dsRNA molecules

(Table 19) Exemplary dsRNA molecules

(Table 20) Exemplary dsRNA molecules

(Table 21) Exemplary dsRNA molecules

(Table 22) Exemplary dsRNA molecules

(Table 23) Exemplary dsRNA molecules

(Table 24) Exemplary dsRNA molecules

(Table 25) Exemplary dsRNA molecules

All United States patents, United States patent application publications, foreign patents, foreign patent applications, and non-patent publications mentioned herein are incorporated by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to take advantage of the ideas of various patents, applications, and publications to yield further embodiments.

These and other changes can be made to the present aspects in light of the above detailed description. In general, the terms used in the appended claims should not be construed to limit the scope of the claims to the specific embodiments disclosed in the specification and claims, but rather to limit the claims to the specific embodiments disclosed in the specification and claims. It is to be interpreted as including all possible embodiments along with the full scope of equivalents permitted in the claims. Therefore, the claims are not limited by this disclosure.

Claims (57)

A dsRNA agent comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides, each nucleotide independently being modified or unmodified;
the sense strand contains a 2'-fluoronucleotide at position 10 counting from the 5' end of the sense strand;
The antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7 and 12 counting from the 5' end of the antisense strand;
(i) the antisense strand contains a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand and a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, or (ii) the antisense strand contains a 2'-deoxynucleotide at position 14 or 16, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand; -contains nucleotides other than fluoronucleotides,
The dsRNA agent. 2. The dsRNA agent of claim 1, wherein the sense strand further comprises a 2'-fluoronucleotide at position 11 counting from the 5' end of the sense strand. 3. The dsRNA agent of claim 1 or 2, wherein the sense strand further comprises a 2'-fluoronucleotide at position 9 counting from the 5' end of the sense strand. dsRNA according to any one of claims 1 to 3, wherein the sense strand further comprises 2'-fluoronucleotides at positions 9 and 11 counting from the 5th end of the sense strand. 5. The dsRNA agent according to any one of claims 1 to 4, wherein the sense strand comprises 2'-fluoronucleotides at positions 8 and 9 counting from the 5th end of the sense strand. 6. The dsRNA agent according to any one of claims 1 to 5, wherein the sense strand comprises 2'-fluoronucleotides at positions 11 and 12 counting from the 5th end of the sense strand. 7. The dsRNA agent according to any one of claims 1 to 6, wherein the sense strand comprises a nucleotide other than 2'-fluoro at position 7 counting from the 5' end of the sense strand. Any of claims 1 to 7, wherein the sense strand comprises 2'-fluoronucleotides at positions 9, 10 and 11 counting from the 5' end of the sense strand, and a nucleotide other than 2'-fluoro at position 7. dsRNA according to item 1. dsRNA agent according to any one of claims 1 to 8, wherein the sense strand comprises at least one 2'-OMe nucleotide. 10. The dsRNA agent according to any one of claims 1 to 9, wherein the sense strand comprises a 2'-OMe nucleotide at position 7 counting from the 5' end of the sense strand. Any one of claims 1 to 10, wherein the sense strand comprises 2'-fluoronucleotides at positions 9, 10 and 11 counting from the 5' end of the sense strand and a 2'-OMe nucleotide at position 7. dsRNA agents as described. the antisense strand comprises a 2'-fluoronucleotide at position 14 of the antisense strand counting from the 5' end of the antisense strand, and a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, counting from the 5' end of the antisense strand; dsRNA agent according to any one of claims 1 to 11. The antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, and 12, a 2'-fluoronucleotide at position 14, and a 2'-fluoronucleotide at position 16, counting from the 5' end of the antisense strand. 13. The dsRNA agent according to any one of claims 1 to 12, comprising nucleotides other than -deoxy or 2'-fluoronucleotides. 14. The dsRNA agent according to any one of claims 1 to 13, wherein the antisense strand comprises a 2'-OMe nucleotide at position 16 counting from the 5' end of the antisense strand. The antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand, a 2'-fluoronucleotide at position 14, and a 2'-fluoronucleotide at position 16. 15. The dsRNA agent according to any one of claims 1 to 14, comprising an OMe nucleotide. The antisense strand contains a 2'-deoxynucleotide at position 14 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand. 12. The dsRNA agent according to any one of claims 1 to 11, comprising nucleotides other than fluoronucleotides. The antisense strand contains a 2'-deoxynucleotide at position 14 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand. 17. The dsRNA agent according to any one of claims 1-11 or 16, comprising an OMe nucleotide. the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, and 14 of the antisense strand counting from the 5' end of the antisense strand; 18. The dsRNA agent according to any one of claims 1-11 or 16-17, comprising a nucleotide other than a 2'-fluoronucleotide at counting position 7. The antisense strand contains a 2'-deoxynucleotide at position 16 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand. 19. The dsRNA agent according to any one of claims 1 to 18, comprising nucleotides other than fluoronucleotides. The antisense strand contains a 2'-deoxynucleotide at position 16 of the antisense strand, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand. 20. The dsRNA agent according to any one of claims 1 to 19, comprising an OMe nucleotide. the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, and 16 of the antisense strand counting from the 5' end of the sense strand; 21. A dsRNA agent according to any one of claims 1 to 20, comprising a nucleotide other than 2'-fluoro in counting position 7. the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, 14, and 16 of the antisense strand counting from the 5' end of the antisense strand; 22. The dsRNA agent according to any one of claims 1-11 or 16-21, comprising a nucleotide other than 2'-fluoro in position 7 counting from the end. The antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12, 14 and 16, and the sense strand contains a 2'-non-fluoro nucleotide at position 7 counting from the 5' end of the sense strand. 23. The dsRNA agent according to any one of claims 1 to 11 or 16 to 22, comprising: the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12 and 16 of the antisense strand, counting from the 5' end of the antisense strand, and a 2'-fluoro at position 14; 21. A dsRNA agent according to any one of claims 1-15 or 19-20, wherein the strand comprises a nucleotide other than 2'-fluoro at position 7 counting from the 5' end of the sense strand. the antisense strand contains 2'-deoxynucleotides at positions 2, 5, 7, 12 and 16 of the antisense strand, counting from the 5' end of the antisense strand, and a 2'-fluoro at position 14; 25. The dsRNA agent according to any one of claims 1-15, 19-20 or 24, wherein the strand comprises a 2'-OMe nucleotide at position 7 counting from the 5' end of the sense strand. 26. The dsRNA agent according to any one of claims 1 to 25, comprising a ligand. 27. The dsRNA agent according to any one of claims 1 to 26, wherein the sense strand comprises a ligand. 28. The dsRNA agent of claim 27, wherein the ligand is at the 3' end of the sense strand. 28. The dsRNA agent of claim 27, wherein the ligand is at the 5' end of the sense strand. 30. The dsRNA agent according to any one of claims 26 to 29, wherein the ligand comprises an ASGPR ligand. 30. The dsRNA agent according to any one of claims 26 to 29, wherein the ligand is a lipophilic group. 32. The dsRNA agent of claim 31, wherein the ligand is a C _10-30 aliphatic group. 33. The dsRNA agent of claim 32, wherein the C _10-30 aliphatic group is a C _10-30 alkyl group. 34. The dsRNA agent according to claim 33, wherein the C _10-30 alkyl group is a linear or branched tetradecyl, hexadecyl, octadecyl, icosyl, docosyl or tetracosyl group. 28. The dsRNA agent of claim 27, wherein the ligand is conjugated to a non-terminal nucleotide of the sense strand. the ligand is conjugated to the 2' position of the non-terminal nucleotide of the sense strand and optionally to one of positions 5, 6, 7 or 8 of the sense strand counting from the 5' end; 36. The dsRNA agent according to claim 35. the ligand comprises an abasic nucleotide, optionally the abasic nucleotide being an inverted nucleotide, linked to the strand of the dsRNA agent via a 5'→5' linkage or a 3'→3' linkage; 37. The dsRNA agent according to any one of claims 26 to 36. 38. A dsRNA agent according to any one of claims 26 to 37, wherein the ligand is attached to the 3' end of the sense strand. 39. The dsRNA agent of claim 38, wherein the ligand is attached to the 3' end of the sense strand via a 3'→3' linkage. 40. A dsRNA agent according to any one of claims 1 to 39, wherein the dsRNA comprises two ligands. 41. The dsRNA of claim 40, wherein the sense strand comprises a first ligand attached to the 3' end of the sense strand and a second ligand attached to the 5' end of the sense strand. the first ligand comprises an abasic nucleotide, the second ligand comprises an ASGPR ligand, and optionally the abasic nucleotide is an inverted nucleotide linked to the sense strand via a 3'→3' linkage. , dsRNA according to claim 41. 43. The dsRNA agent of any one of claims 1-42, comprising at least two phosphorothioate internucleotide linkages. 44. The dsRNA agent of any one of claims 1-43, wherein the sense strand comprises at least two phosphorothioate internucleotide linkages between the first five nucleotides counting from the 5' end of the sense strand. the antisense strand contains at least two phosphorothioate internucleotide linkages between the first five nucleotides counted from the 5' end of the antisense strand, and between the first five nucleotides counted from the 3' end of the antisense strand; 45. The dsRNA agent of any one of claims 1-44, wherein the dsRNA agent comprises at least two phosphorothioate internucleotide linkages. 46. The dsRNA agent of any one of claims 1-45, wherein the dsRNA has a double-stranded region of 18 to about 25 base pairs. 47. The dsRNA agent according to any one of claims 1 to 46, wherein the sense strand is 18 to 23 nucleotides in length. 48. The dsRNA agent according to any one of claims 1 to 47, wherein the antisense strand is 18 to 25 nucleotides in length. A dsRNA agent comprising a sense strand and an antisense strand, the sense strand being 18 to 23 nucleotides in length and containing a 2'-fluoronucleotide at position 10 counting from the 5' end of the sense strand; contains a 2'-fluoronucleotide at position 9 or 11, counting from the 5' end, and the antisense strand is 18 to 25 nucleotides long, at positions 2, 5, 7 and 11, counting from the 5' end of the antisense strand. 12 contains a 2'-deoxynucleotide,
(i) the antisense strand contains a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand and a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, or (ii) the antisense strand contains a 2'-deoxynucleotide at position 14 or 16, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand; -contains nucleotides other than fluoronucleotides,
The dsRNA agent. A dsRNA agent comprising a sense strand and an antisense strand, the sense strand being 18 to 23 nucleotides in length and containing 2'-fluoronucleotides at positions 9, 10 and 11 counting from the 5' end of the sense strand. , and the antisense is 18 to 25 nucleotides in length and contains 2'-deoxynucleotides at positions 2, 5, 7, and 12 counting from the 5' end of the antisense strand;
(i) the antisense strand contains a 2'-fluoronucleotide at position 14 counting from the 5' end of the antisense strand and a nucleotide other than a 2'-deoxy or 2'-fluoronucleotide at position 16, or (ii) the antisense strand contains a 2'-deoxynucleotide at position 14 or 16, counting from the 5' end of the antisense strand, and the sense strand contains a 2'-deoxynucleotide at position 7, counting from the 5' end of the sense strand; -contains nucleotides other than fluoronucleotides,
The dsRNA agent. 51. The dsRNA agent according to any one of claims 1 to 50, comprising a phosphate mimic at the 5' end of the antisense strand. 52. The dsRNA agent of claim 51, wherein the phosphate mimic is 5'-E-vinylphosphonate. Phosphate mimic has the structure:

53. The dsRNA agent according to claim 52, wherein the dsRNA agent is a 5'-cyclopropylphosphonate having the formula: where * is a bond to the C5 position of the nucleotide at the 5' end. The remaining nucleotides in the sense strand (i.e., those in positions not otherwise defined) are unmodified nucleotides or modified nucleotides, optionally 2'-OMe, 2'-F, 2'-H and 2'-OC _10-30 aliphatic groups, with the proviso that no more than one modified nucleotide is a 2'-OC _10-30 aliphatic group. 53. The dsRNA agent according to any one of 53. The remaining nucleotides in the sense strand (i.e. the nucleotides in positions not otherwise defined) are a group consisting of 2'-OMe, 2'-F, 2'-H and 2'-OC _10-30 aliphatic groups. 55. The dsRNA agent according to any one of claims 1 to 54, wherein no more than one modified nucleotide is a 2'-OC _10-30 aliphatic group. The remaining nucleotides in the antisense strand (i.e., nucleotides at positions not otherwise defined) are unmodified nucleotides or modified nucleotides, optionally 2'-OMe, 2'-F, 2'- a modified nucleotide selected from the group consisting of 56. The dsRNA agent according to any one of claims 1 to 55. The remaining nucleotides in the antisense strand (i.e., nucleotides at positions not otherwise defined) are selected from the group consisting of 2'-OMe, 2'-F, 2'-H, GNA and 3'-RNA. 57. Modified nucleotides according to any one of claims 1 to 56, wherein the 3'-RNA is optionally 3'-OH, with the proviso that no more than one modified nucleotide is GNA or 3'-RNA. dsRNA agents.

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